JP7109613B2 - Anti-CD22 antibody-maytansine conjugates and methods of use thereof - Google Patents

Description

The present invention relates to anti-CD22 antibody-maytansine conjugates and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS This application is based on the 35 U . S. C.
. It claims benefits under §119(e).

The field of protein-small molecule therapeutic conjugates has advanced significantly, providing a large number of clinically beneficial drugs, and expected to provide many more in the coming years. Protein conjugate therapeutics can offer several advantages that lead to reduced side effects, for example, due to specificity, multiple functions and relatively low off-target activity. Chemical modification of proteins can extend these advantages by making them more potent, stable, or versatile.

A number of standard chemical transformations are commonly used to generate and manipulate post-translational modifications on proteins. Numerous methods exist by which the side chains of particular amino acids can be selectively modified. For example, carboxylic acid side chains (aspartate and glutamate) can be targeted by initial activation with water-soluble carbodiimide reagents and then subjected to reaction with amines.
Similarly, lysines can be targeted through the use of activated esters or isothiocyanates, and cysteine thiols can be targeted with maleimides and α-halocarbonyls.

One important challenge in creating chemically modified protein therapeutics or reagents is
The goal is to produce such proteins in a biologically active, homogeneous form. Conjugation (binding) of drugs or detectable labels to polypeptides is difficult to control, resulting in heterogeneous mixtures of conjugates that differ in the number of attached drug molecules and the location of chemical conjugation. In some cases, means of synthetic organic chemistry are used to guide the precise and selective formation of chemical bonds on the polypeptide to determine the site of conjugation and/or the drug or detectable conjugated to the polypeptide. It would be desirable to control the label.

General The present disclosure provides anti-CD22 antibody-maytansine conjugate constructs. The disclosure also includes methods of making such conjugates and methods of using them.

Some aspects of the disclosure are represented by formula (I):

[In the formula,
Z is ^CR4 or N;
R ¹ is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
selected from substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl;
R ² and R ³ are each independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acylamino, selected from aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, or ^R2 and R ³ may optionally be cyclically linked to form a 5- or 6-membered heterocyclyl;
Each ^R4 is independently hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acylamino,
selected from aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl;
L is −(T ¹ −V ¹ ) _a −(T ² −V ² ) _b −(T ³ −V ³ ) _c −(T ⁴ −V ⁴ )
_d -comprising linkers, where a, b, c and d are each independently 0 or 1 and the sum of a, b, c and d is 1-4;
T ¹ , T ² , T ³ and T ⁴ are each independently (C ₁ -C ₁₂ )alkyl, substituted (C ₁ -C ₁₂ )alkyl, (EDA) _w , (PEG) _n , (AA) _p , −(CR ¹³
OH) selected from _h- , piperidine-4-amino (4AP), acetal groups, hydrazines, disulfides and esters, where EDA is an ethylenediamine moiety, PE
G is polyethylene glycol or modified polyethylene glycol, AA is an amino acid residue, w is an integer from 1 to 20, n is an integer from 1 to 30, p is an integer from 1 to 20, h is an integer from 1 to 12;
V ¹ , V ² , V ³ and V ⁴ are each independently a covalent bond, -CO-, -NR ^1
5 -, -NR ¹⁵ (CH ₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -N
R ¹⁵ CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —S
selected from the group consisting of O ₂ —, —SO ₂ NR ¹⁵ —, —NR ¹⁵ SO ₂ — and —P(O)OH—, wherein q is an integer from 1 to 6;
each R ¹³ is independently selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl;
Each R ¹⁵ is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cyclo selected from alkyl, heterocyclyl and substituted heterocyclyl;
W1 is ^a maytansinoid; and W2 is an anti ^- CD22 antibody], comprising at least one modified amino acid residue.

In some embodiments,
T ¹ is selected from (C ₁ -C ₁₂ )alkyl and substituted (C ₁ -C ₁₂ )alkyl;
T ² , T ³ and T ⁴ are each independently (EDA) _w , (PEG) _n , (C ₁
—C ₁₂ )alkyl, substituted (C ₁ -C ₁₂ )alkyl, (AA) _p , —(CR ¹³ OH)
_h -, piperidine-4-amino (4AP), acetal groups, hydrazines and esters; and V ¹ , V ² , V ³ and V ⁴ are each independently a covalent bond, -CO-, - NR ^1
5 -, -NR ¹⁵ (CH ₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -N
R ¹⁵ CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —S
selected from the group consisting of O ₂ —, —SO ₂ NR ¹⁵ —, —NR ¹⁵ SO ₂ — and —P(O)OH—;
here,
(PEG) _n is

where n is an integer from 1 to 30;
EDA has the following structure:

wherein y is an integer from 1 to 6 and r is 0 or 1;
Piperidine-4-amino is

is;
each R ¹² and R ¹⁵ is independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moieties, aryl and substituted aryl, wherein any two adjacent R ¹² groups are cyclically linked; optionally forming a piperazinyl ring; and R ¹³ is selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl.

In some embodiments, T ¹ , T ² , T ³ and T ⁴ and V ¹ , V ² , V ³ and V ⁴ are selected from the table below.

In some embodiments, L is selected from one of the following structures.

In the above formula,
each f is independently 0 or an integer from 1 to 12;
each y is independently 0 or an integer from 1 to 20;
each n is independently 0 or an integer from 1 to 30;
each p is independently 0 or an integer from 1 to 20;
each h is independently 0 or an integer from 1 to 12;
Each R is independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy,
is aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl; and each R' is independently H, a side chain group of an amino acid, alkyl, substituted alkyl, alkenyl , substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl.

In certain embodiments, the maytansinoid has the formula:

belongs to.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is 4AP, V ² is -CO-, T ³ is (C ₁ -C ₁₂ )alkyl, V ³ is —CO—, T ⁴ is absent and V ⁴ is absent.

In some embodiments, the linker L has the structure:

(wherein each f is independently an integer from 1 to 12 and n is an integer from 1 to 30).

In certain embodiments, the anti-CD22 antibody comprises amino acids 1-847 (amino acids 1-847), amino acids 1-759 (amino acids 1-759), amino acids 1-751 of the CD22 amino acid sequences shown in FIGS. (amino acids 1-751) or amino acids 1-670
Binds an epitope at (amino acids 1-670).

In certain embodiments, the anti-CD22 antibody has formula (II):
X ¹ (FGly') X ² Z ²⁰ X ³ Z ³⁰ (II)
(In the formula,
FGly' is a modified amino acid residue of formula (I);
^Z20 is a proline or alanine residue;
Z ³⁰ is a basic amino acid or an aliphatic amino acid;
X ¹ may or may not be present, and if present may be ^any amino acid, but is present if said sequence is at the N-terminus of said conjugate; and X2 and X3 ^{are each} independently any amino acid)
contains an array of

In some embodiments, the sequence is L(FGly')TPSR.

In some embodiments, Z ³⁰ is selected from R, K, H, A, G, L, V, I and P, X ¹ is selected from L, M, S and V, X ² and X ³ are , each independently,
selected from S, T, A, V, G and C;

In certain embodiments, the modified amino acid residue is located at the C-terminus of the heavy chain constant region of the anti-CD22 antibody.

In certain embodiments, the heavy chain constant region has formula (II):
X ¹ (FGly') X ² Z ²⁰ X ³ Z ³⁰ (II)
(In the formula,
FGly' is a modified amino acid residue of formula (I);
^Z20 is a proline or alanine residue;
Z ³⁰ is a basic amino acid or an aliphatic amino acid;
X ¹ may or may not be present, and if present may be ^any amino acid, but is present if said sequence is at the N-terminus of said conjugate; and X2 and X3 ^{are each} independently any amino acid)
wherein the sequence is C-terminal to the amino acid sequence SLSLSPG.

In certain embodiments, the heavy chain constant region has the sequence SPGSL(FGly')TPSRG
Including S.

In some embodiments, Z ³⁰ is selected from R, K, H, A, G, L, V, I and P, X ¹ is selected from L, M, S and V, X ² and X ³ are , each independently,
selected from S, T, A, V, G and C;

In certain embodiments, the modified amino acid residue is located within the light chain constant region of the anti-CD22 antibody.

In certain embodiments, the light chain constant region has formula (II):
X ¹ (FGly') X ² Z ²⁰ X ³ Z ³⁰ (II)
(In the formula,
FGly' is a modified amino acid residue of formula (I);
^Z20 is a proline or alanine residue;
Z ³⁰ is a basic amino acid or an aliphatic amino acid;
X ¹ may or may not be present, and if present may be ^any amino acid, but is present if said sequence is at the N-terminus of said conjugate; and X2 and X3 ^{are each} independently any amino acid)
wherein said sequence is present at the C-terminus of the sequence KVDNAL and/or present at the N-terminus of the sequence QSGNSQ.

In some embodiments, the light chain constant region has the sequence KVDNAL(FGly')TPSR
Includes QSGNSQ.

In some embodiments, Z ³⁰ is selected from R, K, H, A, G, L, V, I and P, X ¹ is selected from L, M, S and V, X ² and X ³ are , each independently,
selected from S, T, A, V, G and C;

In certain embodiments, the modified amino acid residue is located within the heavy chain CH1 region of the anti-CD22 antibody.

In certain embodiments, the heavy chain CH1 region has formula (II):
X ¹ (FGly') X ² Z ²⁰ X ³ Z ³⁰ (II)
(In the formula,
FGly' is a modified amino acid residue of formula (I);
^Z20 is a proline or alanine residue;
Z ³⁰ is a basic amino acid or an aliphatic amino acid;
X ¹ may or may not be present, and if present may be ^any amino acid, but is present if said sequence is at the N-terminus of said conjugate; and X2 and X3 ^{are each} independently any amino acid)
wherein said sequence is present at the C-terminus of the sequence SWNSGA and/or present at the N-terminus of the sequence GVHTFP.

In some embodiments, the heavy chain CH1 region has the sequence SWNSGAL(FGly')TP
Includes SRGVHTFP.

In some embodiments, Z ³⁰ is selected from R, K, H, A, G, L, V, I and P, X ¹ is selected from L, M, S and V, X ² and X ³ are , each independently,
selected from S, T, A, V, G and C;

In certain embodiments, the modified amino acid residue is located within the heavy chain CH2 region of the anti-CD22 antibody.

In certain embodiments, the modified amino acid residue is located within the heavy chain CH3 region of the anti-CD22 antibody.

Some aspects of the disclosure include pharmaceutical compositions comprising a conjugate described herein and a pharmaceutically acceptable excipient.

Some aspects of the disclosure include methods comprising administering to a subject an effective amount of a conjugate described herein.

Some aspects of the present disclosure include methods of treating cancer in a subject. The method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate described herein, wherein the administration is effective to treat cancer in the subject. be.

Some aspects of the present disclosure include methods of delivering drugs to target sites in a subject. The method comprises administering to a subject a pharmaceutical composition comprising a conjugate described herein, wherein the administering comprises a therapeutically effective amount of drug from the conjugate at a target site in the subject. is effective to emit

Definitions The following terms have the following meanings unless otherwise indicated. Any terms not defined have their art-recognized meanings.

“Alkyl” is a monovalent saturated aliphatic hydrocarbyl having 1 to 10 carbon atoms, such as 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms means the base. The term includes, for example, straight-chain and branched hydrocarbyl groups such as methyl ( _CH3
-), ethyl (CH ₃ CH ₂ -), n-propyl (CH ₃ CH ₂ CH ₂ -), isopropyl ((CH ₃ ) ₂ CH-), n-butyl (CH ₃ CH ₂ CH ₂ CH ₂ -) , isobutyl (
(CH ₃ ) ₂ CHCH ₂ —), sec-butyl ((CH ₃ )(CH ₃ CH ₂ )CH—),
t-butyl ((CH ₃ ) ₃ C—), n-pentyl (CH ₃ CH ₂ CH ₂ CH ₂ CH ₂ —)
and neopentyl ((CH ₃ ) ₃ CCH ₂ —).

The term "substituted alkyl" means that _one or more carbon atoms (other than the C1 carbon atom) in the alkyl chain are optionally substituted by a heteroatom such as -O-, -N-, -S-, -S(O) _n- (where
n is 0 to 2), -NR- (where R is hydrogen or alkyl), etc., alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,
Cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thio heterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, - SO-heteroaryl, —SO ₂
-alkyl, -SO ₂ -aryl, -SO ₂ -heteroaryl and -NR ^a R ^b (wherein R' and R'' are the same which may or may not be selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocycle), as defined herein. means an alkyl group containing

“Alkylene” includes —O—, —NR ¹⁰ —, —NR ¹⁰ C(O)—, —C(O)NR ¹
1 or more groups selected from ⁰ - may optionally intervene, preferably 1 to 6,
More preferably it means a straight chain or branched divalent aliphatic hydrocarbyl group having 1 to 3 carbon atoms. This term includes, for example, methylene ₍ --CH.sub.2--), ethylene _{( --CH.sub.2CH.sub.2--}
), n-propylene (--CH ₂ CH ₂ CH ₂ --), isopropylene (--CH ₂ CH(CH
₃ )-), (-C(CH ₃ ) ₂ CH ₂ CH ₂ -), (-C(CH ₃ ) ₂ CH ₂ C(O)-
), (—C(CH ₃ ) ₂ CH ₂ C(O)NH—), (—CH(CH ₃ )CH ₂ —), and the like.

"Substituted alkylene" means an alkylene group in which from 1 to 3 hydrogens have been replaced with substituents described for carbons in the definition of "substituted" below.

The term "alkane" means alkyl and alkylene groups as defined herein.

The terms "alkylaminoalkyl", "alkylaminoalkenyl" and "alkylaminoalkynyl" refer to the group R'NHR''-, where R' is an alkyl group as defined herein. and R″ is an alkylene, alkenylene or alkynylene group as defined herein.

The terms "alkaryl" or "aralkyl" refer to the groups -alkylene-aryl and -substituted alkylene-aryl, where alkylene, substituted alkylene and aryl are defined herein.

"Alkoxy" means the group -O-alkyl, where alkyl is as defined herein. Alkoxy is for example methoxy, ethoxy, n-propoxy,
Including isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy and the like. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O- and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl and alkynyl are herein as defined in

The term "substituted alkoxy" refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O- and substituted alkynyl-O-, where substituted alkyl, Substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

The term "alkoxyamino" refers to the group -NH-alkoxy, where alkoxy is defined herein.

The term "haloalkoxy" means an alkyl-O- group in which one or more hydrogen atoms on the alkyl group have been replaced with a halo group, and includes groups such as trifluoromethoxy and the like.

The term "haloalkyl" means a substituted alkyl group as described above in which one or more hydrogen atoms on the alkyl group have been replaced with a halo group. Examples of such groups include, but are not limited to, fluoroalkyl groups such as trifluoromethyl, difluoromethyl, trifluoroethyl, and the like.

The term "alkylalkoxy" refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl and substituted alkylene-O-substituted alkyl, where alkyl, substituted alkyl, Alkylene and substituted alkylene are as defined herein.

The term "alkylthioalkoxy" means -alkylene-S-alkyl, alkylene-S
-substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substituted alkyl, where alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

"Alkenyl" is a straight or branched hydrocarbyl having 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, and having at least 1 and preferably 1 to 2 sites of double bond unsaturation. means the base. This term includes, for example, bi-vinyl, allyl and but-3-ene-1
- including files. The term includes cis and trans isomers or mixtures of these isomers.

The term "substituted alkenyl" includes alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -
1 to 5 substituents or 1 to 3 selected from SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl and -SO ₂ -heteroaryl means an alkenyl group as defined herein having one substituent.

"Alkynyl" means a straight or branched chain having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and having at least 1 and preferably 1 to 2 sites of triple bond unsaturation. means a valent hydrocarbyl group. Examples of such alkynyl groups include acetylenyl (C≡CH)
and propargyl (-CH ₂ C≡CH).

The term "substituted alkynyl" includes alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -
1 to 5 substituents or 1 to 3 selected from SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl and -SO ₂ -heteroaryl means an alkynyl group as defined herein having one substituent.

"Alkynyloxy" means -O-, where alkynyl is as defined herein
means an alkynyl group. Alkynyloxy includes, for example, ethynyloxy, propynyloxy, and the like.

"Acyl" refers to HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)- )-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-
, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(
O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)- and substituted heterocyclyl-C(O)- where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle (group ) and substituted heterocycle (group) are as defined herein. For example, acyl includes the “acetyl” group CH ₃ C(O)—.

"Acylamino" means -NR ²⁰ C(O)alkyl, -NR ²⁰ C(O) substituted alkyl,
NR ²⁰ C(O) cycloalkyl, —NR ²⁰ C(O) substituted cycloalkyl, —NR ²⁰
C(O) cycloalkenyl, —NR ²⁰ C(O) substituted cycloalkenyl, —NR ²⁰ C(
O) alkenyl, —NR ²⁰ C(O) substituted alkenyl, —NR ²⁰ C(O) alkynyl,
—NR ²⁰ C(O) substituted alkynyl, —NR ²⁰ C(O) aryl, —NR ²⁰ C(O)
means the groups substituted aryl, —NR ²⁰ C(O)heteroaryl, —NR ²⁰ C(O) substituted heteroaryl, —NR ²⁰ C(O) heterocycle and —NR ²⁰ C(O) substituted heterocycle;
wherein R ²⁰ is hydrogen or alkyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted Heteroaryl, heterocycle(s) and substituted heterocycle(s) are as defined herein.

The term “aminocarbonyl” or “aminoacyl” refers to the group —C(O)NR ²¹ R ²² where R ²¹ and R ²² are independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted selected from the group consisting of alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocycle (group) and substituted heterocycle (group) optionally, R ²¹ and R ²² may be linked together with the nitrogen attached to them to form a heterocyclic or substituted heterocyclic group, alkyl, substituted alkyl,
Alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle (group) and substituted heterocycle (group) are As defined in the specification.

“Aminocarbonylamino” means the group —NR ²¹ C(O)NR ²² R ²³ where R ²¹ , R ²² and R ²³ are independently selected from hydrogen, alkyl, aryl or cycloalkyl or two R groups are joined to form a heterocyclyl group.

The term "alkoxycarbonylamino" refers to the group -NRC(O)OR, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl or heterocyclyl, wherein Alkyl, substituted alkyl, aryl, heteroaryl and heterocyclyl are as defined herein.

The term "acyloxy" includes alkyl-C(O)O-, substituted alkyl-C(O)O-,
Cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—, aryl-C(O
)O—, substituted aryl-C(O)O— and heterocyclyl-C(O)O—, where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl and heterocyclyl are As defined herein.

" _{Aminosulfonyl} " means the group ^-SO2NR21R22 , where ^R21 and ^R22 are independently ^hydrogen , alkyl, substituted alkyl, alkenyl, substituted alkenyl,
selected from the group consisting of alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic group, substituted heterocyclic group, optionally R ²¹ and ^R22
may be joined together with the nitrogen to which they are attached to form a heterocyclic or substituted heterocyclic group, including alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted Cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle(s) and substituted heterocycle(s) are as defined herein.

" ^{Sulfonylamino} " means the group ^-NR21SO2R22 , where ^R21 and ^R22 _are independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
selected from the group consisting of alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic group and substituted heterocyclic group; ²¹ and R
²² may be linked together with the nitrogen attached to them to form a heterocyclic or substituted heterocyclic group, which includes alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, Substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle(s) and substituted heterocycle(s) are as defined herein.

"Aryl" or "Ar" means a ring system having a single ring (e.g., in a phenyl group) or multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl and indanyl) means a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a fused ring which may or may not be aromatic, provided that the point of attachment is through an atom of the aromatic ring is. This term includes, for example, phenyl and naphthyl, by way of example. Unless otherwise limited by the definition for an aryl substituent, such aryl groups can optionally be acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl,
heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -
1-5 substituents selected from SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl, -SO ₂ -heteroaryl and trihalomethyl or It may be substituted with a substituent.

"Aryloxy" means the group -O-aryl, where aryl is as defined herein, including, for example, phenoxy, naphthoxy and the like, optionally substituted aryl groups (also as defined herein).

"Amino" means the group _-NH2 .

The term "substituted amino" refers to the group -NRR, where each R is independently hydrogen,
selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclyl, provided that at least one R is hydrogen is not.

The term "azido" means the group _-N3 .

"Carboxyl", "carboxy" or "carboxylate" means _-CO2H or a salt thereof.

The term "carboxyester" or "carboxyester" or "carboxyalkyl" or "carboxyalkyl" refers to -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl , —C(O)O-substituted alkenyl, —C(O)O—
alkynyl, —C(O)O-substituted alkynyl, —C(O)O-aryl, —C(O)O—
substituted aryl, —C(O)O-cycloalkyl, —C(O)O-substituted cycloalkyl, —
C(O)O-cycloalkenyl, —C(O)O-substituted cycloalkenyl, —C(O)O—
heteroaryl, —C(O)O-substituted heteroaryl, —C(O)O-heterocycle and —C
(O) means the group O-substituted heterocycle, wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,
Substituted heteroaryl, heterocycle(s) and substituted heterocycle(s) are as defined herein.

"(Carboxyester)oxy" or "carbonate" means -O-C(O)O-alkyl, -O-C(O)O-substituted alkyl, -O-C(O)O-alkenyl, -O-C (O
) O-substituted alkenyl, -OC(O)O-alkynyl, -OC(O)O-substituted alkynyl, -OC(O)O-aryl, -OC(O)O-substituted Aryl, —O—C(O)
O-cycloalkyl, -O-C(O)O-substituted cycloalkyl, -O-C(O)O-cycloalkenyl, -O-C(O)O-substituted cycloalkenyl, -O-C(O) O-heteroaryl, -O-C(O)O-substituted heteroaryl, -O-C(O)O-heterocycle and -O
means the group —C(O)O-substituted heterocycle, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, Substituted aryl, heteroaryl, substituted heteroaryl, heterocycle(s) and substituted heterocycle(s) are as defined herein.

"Cyano" or "nitrile" means the group -CN.

"Cycloalkyl" means cyclic alkyl groups of from 3 to 10 carbon atoms having mono- or polycyclic rings, including fused, bridged and spiro ring systems. Examples of suitable cycloalkyl groups include, eg, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. Such cycloalkyl groups include, for example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc., or polycyclic structures such as adamantanyl.

The term "substituted cycloalkyl" includes alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxy aminoacyl, azide, cyano, halogen, hydroxyl, oxo,
thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino , alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO ₂ -alkyl, —SO ₂ -substituted alkyl, —SO ₂ -aryl and —SO ₂ - means a cycloalkyl group having 1 to 5 substituents or 1 to 3 substituents selected from heteroaryl.

"Cycloalkenyl" means a non-aromatic cyclic alkyl group of 3-10 carbon atoms having a single ring or multiple rings and at least one double bond, preferably 1-2 double bonds. means

The term "substituted cycloalkenyl" includes alkoxy, substituted alkoxy, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,
carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, from nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO ₂ -alkyl, —SO ₂ -substituted alkyl, —SO ₂ -aryl and —SO ₂ -heteroaryl It means a cycloalkenyl group having 1 to 5 or 1 to 3 selected substituents.

"Cycloalkynyl" means non-aromatic cycloalkyl groups of 5 to 10 carbon atoms having single or multiple rings and at least one triple bond.

"Cycloalkoxy" means -O-cycloalkyl.

"Cycloalkenyloxy" means -O-cycloalkenyl.

"Halo" or "halogen" means fluoro, chloro, bromo and iodo.

"Hydroxy" or "hydroxyl" means the group -OH.

"Heteroaryl" is an aromatic of 1 to 15 carbon atoms, such as 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in a ring means the base. Such heteroaryl groups may have a single ring (such as pyridinyl, imidazolyl or furyl) or multiple condensed rings (such as in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl) within the ring system. is possible, wherein at least one ring in the ring system is aromatic. To satisfy valence requirements, any heteroatom within such heteroaryl ring may be attached to H or a substituent (e.g., an alkyl group or other substituent as described herein). It does not have to be connected. In certain embodiments, the nitrogen and/or sulfur ring atoms of the heteroaryl group are optionally oxidized to provide N-oxide (N→O), sulfinyl, or sulfonyl moieties. This term includes, for example, pyridinyl, pyrrolyl, indolyl, thiophenyl and furanyl. Unless otherwise limited by the definition for heteroaryl substituents, such heteroaryl groups can optionally be acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted Alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, hetero aryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO 1 to 5 substituents or 1 to 3 substitutions selected from -heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl and -SO ₂ -heteroaryl and trihalomethyl may be substituted with a group.

The term "heteroaralkyl" refers to the group -alkylene-heteroaryl, where alkylene and heteroaryl are defined herein. This term includes, for example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

"Heteroaryloxy" means -O-heteroaryl.

"Heterocycle", "heterocyclic (heterocyclic group)", "heterocycloalkyl" and "heterocyclyl" are fused bridged and spiro- It means a saturated or unsaturated group having a single ring or multiple condensed rings, including ring systems. These ring atoms are selected from nitrogen, sulfur or oxygen, wherein in fused ring systems one or more of the rings can be cycloalkyl, aryl or heteroaryl, provided that
The point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atoms of the heteroaryl group are optionally represented by N-oxide, --S(O)-- or --SO ₂
- may be oxidized to give moieties. To satisfy valence requirements, any heteroatom in such heterocycles may be replaced by one or more H or one or more substituents (e.g., alkyl groups or other substituents described herein). It can be bound or unbound.

Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine. , isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine,
indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,
7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also called thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, etc. is included.

Unless otherwise limited by the definition for heterocyclic substituents, such heterocyclic groups are:
optionally alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,
substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl,
thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol,
thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -
1 to 5 or 1 to 3 selected from SO-heteroaryl, —SO ₂ -alkyl, —SO ₂ -substituted alkyl, —SO ₂ -aryl, —SO ₂ -heteroaryl and fused heterocycle;
may be substituted with one substituent.

"Heterocyclyloxy" means the term -O-heterocyclyl.

The term "heterocyclylthio" refers to the group heterocycle-S-.

The term "heterocyclene" means a diradical group formed from a heterocycle as defined herein.

The term "hydroxyamino" means the group -NHOH.

"Nitro" means the group _-NO2 .

"Oxo" means an atom (=O).

“Sulfonyl” means SO ₂ -alkyl, SO ₂ -substituted alkyl, SO ₂ -alkenyl, S
O ₂ -substituted alkenyl, SO ₂ -cycloalkyl, SO ₂ -substituted cycloalkyl, SO ₂
-cycloalkenyl, SO ₂ -substituted cycloalkenyl, SO ₂ -aryl, SO ₂ -substituted aryl, SO ₂ -heteroaryl, SO ₂ -substituted heteroaryl, SO ₂ -heterocycle and SO ₂ -substituted heterocycle; means where alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,
Substituted heteroaryl, heterocycle(s) and substituted heterocycle(s) are as defined herein. Sulfonyl is, for example, methyl-SO ₂ -, phenyl-SO ₂ - and 4
-methylphenyl-SO ₂ -.

"Sulfonyloxy" means -OSO ₂ -alkyl, OSO ₂ -substituted alkyl, OSO ₂ -
alkenyl, OSO ₂ -substituted alkenyl, OSO ₂ -cycloalkyl, OSO ₂ -substituted cycloalkyl, OSO ₂ -cycloalkenyl, OSO ₂ -substituted cycloalkenyl, OSO
means the groups ₂ -aryl, OSO ₂ -substituted aryl, OSO ₂ -heteroaryl, OSO ₂ -substituted heteroaryl, OSO ₂ -heterocycle and OSO ₂ -substituted heterocycle, wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
Cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle(s) and substituted heterocycle(s) are as defined herein .

The term "aminocarbonyloxy" refers to the group -OC(O)NRR, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or a heterocyclic group, wherein and alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic groups are as defined herein.

"Thiol" means the group -SH.

The term "thioxo" or "thioketo" means the atom (=S).

The term "alkylthio" or "thioalkoxy" means the group -S-alkyl
where alkyl is as defined herein. In some embodiments,
The sulfur can be oxidized to -S(O)-. The sulfoxide can exist as one or more stereoisomers.

The term "substituted thioalkoxy" refers to the group -S-substituted alkyl.

The term "thioaryloxy" means the group aryl-S-, where the aryl group is as defined herein and optionally substituted as also defined herein. It includes an aryl group that may be

The term "thioheteroaryloxy" refers to the group heteroaryl-S-, where the heteroaryl group is as defined herein and the desired heteroaryl group is also defined herein. Including aryl groups optionally substituted by

The term "thioheterocyclooxy" refers to the group heterocyclyl-S-, wherein
Heterocyclyl groups are as defined herein and include optionally substituted heterocyclyl groups also as defined herein.

Further to the disclosure herein, the term "substituted" when used to modify a specified group or radical means that one or more hydrogen atoms of that specified group or radical are , each independently of the other, means substituted with the same or different substituents as defined below.

In addition to the groups disclosed for individual terms herein, substituents for replacing one or more hydrogens on a saturated carbon atom in a specified group or radical (any two hydrogens on a single carbon =O, =NR ⁷⁰ , =N-OR ⁷⁰ , =N ₂ or =S) are -R ⁶⁰ , halo, =O, -OR ⁷⁰ , -SR ⁷⁰ unless otherwise indicated , -NR ^8
0 R ⁸⁰ , trihalomethyl, —CN, —OCN, —SCN, —NO, —NO ₂ ,=N ₂ ,
—N ₃ , —SO ₂ R ⁷⁰ , —SO ₂ O ⁻ M ⁺ , —SO ₂ OR ⁷⁰ , —OSO ₂ R ⁷⁰ , —
OSO ₂ O ⁻ M ⁺ , —OSO ₂ OR ⁷⁰ , —P(O)(O ⁻ ) ₂ (M ⁺ ) ₂ , —P(O)
(OR ⁷⁰ )O ⁻ M ⁺ , —P(O)(OR ⁷⁰ ) ₂ , —C(O)R ⁷⁰ , —C(S)R ^7
0 , —C(NR ⁷⁰ )R ⁷⁰ , —C(O)O ⁻ M ⁺ , —C(O)OR ⁷⁰ , —C(S)O
R ⁷⁰ , —C(O)NR ⁸⁰ R ⁸⁰ , —C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ , —OC(O)R
⁷⁰ , —OC(S)R ⁷⁰ , —OC(O)O ⁻ M ⁺ , —OC(O)OR ⁷⁰ , —OC(S
) OR ⁷⁰ , —NR ⁷⁰ C(O)R ⁷⁰ , —NR ⁷⁰ C(S)R ⁷⁰ , —NR ⁷⁰ CO ₂
^- M ⁺ , -NR ⁷⁰ CO ₂ R ⁷⁰ , -NR ⁷⁰ C(S)OR ⁷⁰ , -NR ⁷⁰ C(O)N
R ⁸⁰ R ⁸⁰ , —NR ⁷⁰ C(NR ⁷⁰ )R ⁷⁰ and —NR ⁷⁰ C(NR ⁷⁰ )NR ^8
0 R ⁸⁰ where R ⁶⁰ is optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl,
selected from the group consisting of aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R ⁷⁰ is independently hydrogen or R ⁶⁰ and each R ⁸⁰ is independently R ⁷⁰ , or Two R ⁸⁰ , together with the nitrogen atom to which they are attached, are a 5-, 6- or 7-membered heterocycloalkyl (which optionally is a 1- to 7-membered heterocycloalkyl selected from the group consisting of O, N and S). may contain four identical or different additional heteroatoms, where N may have —H or C ₁ -C ₃ alkyl substituents), and each M ⁺ is A counterion with a single net positive charge. Each M ⁺ is independently, for example, an alkali ion, such as K ⁺ , Na ⁺ , Li ⁺ ; an ammonium ion, such as ⁺ N ^(R60 ) ₄
or alkaline earth ions such as [Ca ²⁺ ] _0.5 , [Mg ²⁺ ] _0.5 or [
Ba ²⁺ ] _0.5 (the subscript “0.5” indicates that one of the counterions of such divalent alkaline earth ion can be the ionized form of the compound of the invention, the other or that the two ionized compounds disclosed herein can serve as counterions for such divalent alkaline earth ions, or (meaning that the doubly ionized compounds of the present invention can serve as counterions for such divalent alkaline earth ions). As specific examples, -NR ⁸⁰ R ⁸⁰ is meant to include -NH ₂ , -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl and N-morpholinyl.

In addition to the disclosure herein, the substituents for hydrogen on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are —R 60 , —R ⁶⁰ , unless otherwise indicated.
halo, -O ^- M ⁺ , -OR ⁷⁰ , -SR ⁷⁰ , -S ^- M ⁺ , -NR ⁸⁰ R ⁸⁰ , trihalomethyl, -CF ₃ , -CN, -OCN, -SCN, -NO, -NO ₂ , —N ₃ , —SO ₂
R ⁷⁰ , —SO ₃ ⁻ M ⁺ , —SO ₃ R ⁷⁰ , —OSO ₂ R ⁷⁰ , —OSO ₃ ⁻ M ⁺ , —O
SO ₃ R ⁷⁰ , -PO ₃ ^-2 (M ⁺ ) ₂ , -P(O)(OR ⁷⁰ )O ^- M ⁺ , -P(O)
(OR ⁷⁰ ) ₂ , —C(O)R ⁷⁰ , —C(S)R ⁷⁰ , —C(NR ⁷⁰ )R ⁷⁰ , —C
O ₂ ⁻ M ⁺ , —CO ₂ R ⁷⁰ , —C(S)OR ⁷⁰ , —C(O)NR ⁸⁰ R ⁸⁰ , —C(
NR ⁷⁰ ) NR ⁸⁰ R ⁸⁰ , —OC(O)R ⁷⁰ , —OC(S)R ^{70 ,} —OCO ₂ -M
⁺ , —OCO ₂ R ⁷⁰ , —OC(S)OR ⁷⁰ , —NR ⁷⁰ C(O)R ⁷⁰ , —NR ⁷⁰
C(S)R ⁷⁰ , —NR ⁷⁰ CO ₂ ⁻ M ⁺ , —NR ⁷⁰ CO ₂ R ⁷⁰ , —NR ⁷⁰ C(S
)OR ⁷⁰ , —NR ⁷⁰ C(O)NR ⁸⁰ R ⁸⁰ , —NR ⁷⁰ C(NR ⁷⁰ )R ⁷⁰ and —NR ⁷⁰ C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ where R ⁶⁰ , R ⁷⁰ , R ⁸⁰ and M ⁺ are as previously defined with the proviso that in the case of substituted alkenes or alkynes the substituent is not —O ⁻ M ⁺ , —OR ⁷⁰ , —SR ⁷⁰ or —S ⁻ M ⁺ .

In addition to the groups disclosed for individual terms herein, substituents for hydrogen on the nitrogen atom in “substituted” heteroalkyl and cycloheteroalkyl groups are —R ⁶⁰ , — O ⁻ M ⁺ , −OR ⁷⁰ , −SR ⁷⁰ , −S ⁻ M ⁺ , −NR ⁸⁰ R
⁸⁰ , trihalomethyl, —CF ₃ , —CN, —NO, —NO ₂ , —S(O) ₂ R ⁷⁰ , —
S(O) ₂ O ⁻ M ⁺ , —S(O) ₂ OR ⁷⁰ , —OS(O) ₂ R ⁷⁰ , —OS(O) ₂ O
⁻ M ⁺ , −OS(O) ₂ OR ⁷⁰ , −P(O)(O ⁻ ) ₂ (M ⁺ ) ₂ , −P(O)(OR
⁷⁰ ) O ⁻ M ⁺ , —P(O)(OR ⁷⁰ )(OR ⁷⁰ ), —C(O)R ⁷⁰ , —C(S)
R ⁷⁰ , —C(NR ⁷⁰ )R ⁷⁰ , —C(O)OR ⁷⁰ , —C(S)OR ⁷⁰ , —C(O
)NR ⁸⁰ R ⁸⁰ , —C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ , —OC(O)R ⁷⁰ , —OC(S
)R ⁷⁰ , —OC(O)OR ⁷⁰ , —OC(S)OR ⁷⁰ , —NR ⁷⁰ C(O)R ⁷⁰ ,
—NR ⁷⁰ C(S)R ⁷⁰ , —NR ⁷⁰ C(O)OR ⁷⁰ , —NR ⁷⁰ C(S)OR ⁷⁰
, —NR ⁷⁰ C(O)NR ⁸⁰ R ⁸⁰ , —NR ⁷⁰ C(NR ⁷⁰ )R ⁷⁰ and —NR ^7
0 C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ where R ⁶⁰ , R ⁷⁰ , R ⁸⁰ and M ⁺ are as previously defined.

Further to the disclosure herein, in some embodiments, the substituted groups are 1, 2, 3
or 4 substituents, 1, 2 or 3 substituents, 1 or 2 substituents, or 1 substituent.

In all of the substituents defined above, the polymer obtained by defining a substituent with further substituents on itself (e.g., a substituted aryl group itself with a substituted aryl group, such as further substituted with a substituted aryl group) It is understood that substituted aryl) having substituted aryl groups as substituted substituents are not intended to be included in the present invention. In such cases, the maximum number of such replacements is three. For example, serial substitutions of substituted aryl groups specifically contemplated in this invention are limited to substituted aryl-(substituted aryl)-substituted aryl.

Unless otherwise indicated, nomenclature of substituents not explicitly defined herein is made by designating the terminal portion of the functional group followed by the adjacent functional group on the side of the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

For any of the groups disclosed herein that contain one or more substituents, of course, such groups are not subject to any sterically impossible and/or synthetically impracticable substitution or substitution. It is also understood not to include patterns. Alternatively, the subject compounds include all stereochemical isomers resulting from substitution of these compounds.

The term "pharmaceutically acceptable salt" means a salt that is acceptable for administration to a patient, e.g., a mammal (salts containing counterions that exhibit acceptable mammalian safety for a given dosage regimen). do. Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" means a pharmaceutically acceptable salt of a compound derived from a variety of organic and inorganic counterions well known in the art and by way of example only, sodium , potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and if the molecule contains a basic functional group, a salt of an organic or inorganic acid such as hydrochloride, hydrobromide, formate, Includes tartrates, besylates, mesylates, acetates, maleates, oxalates, and the like.

The term "salt thereof" means a compound formed when a proton of an acid is replaced by a cation such as a metal cation or an organic cation. Where applicable, the salts are pharmaceutically acceptable salts. However, this is not required for salts of intermediate compounds that are not intended for administration to a patient. For example, salts of the compounds include those in which the compound is protonated with an inorganic or organic acid to form a cation and the conjugate base of the inorganic or organic acid is present as the anionic component of the salt.

"Solvate" means a complex formed by the combination of solvent molecules and solute molecules or ions. Solvents can be organic compounds, inorganic compounds, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide and water. When the solvent is water the solvate formed is a hydrate.

"Stereoisomer" means compounds that have the same atomic connectivity but different arrangements of the atoms in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers and diastereomers.

"Tautomer" means alternate forms of molecules that differ only in the positions of the electron bonds and/or protons of the atoms, such as enol-keto and imine-enamine tautomers, or It includes tautomers of heteroaryl groups containing N=C(H)-NH- ring atom configurations such as pyrazole, imidazole, benzimidazole, triazole and tetrazole. Those skilled in the art will recognize that other tautomeric ring atom arrangements are possible.

The term "or salts or solvates or stereoisomers thereof" refers to all salts, solvates and stereoisomeric permutations, such as pharmaceutically acceptable salts of stereoisomers of a subject compound. It is understood that it is intended to include solvates.

"Pharmaceutically effective amount" and "therapeutically effective amount" are sufficient to treat and/or prevent the occurrence of one or more of the identified disorders or diseases or symptoms thereof. It means the amount of compound. For tumorigenic proliferative disorders, a pharmaceutically or therapeutically effective amount includes, inter alia, amounts sufficient to cause tumor regression or decrease the rate of tumor growth.

"Patient" means human and non-human subjects, particularly mammalian subjects.

The term "treating" or "treatment" as used herein means treating a disease or medical condition in a patient, e.g., a mammal (particularly a human), or such treatment, (a) prevention of the occurrence of a disease or medical condition, e.g., prophylactic treatment in a subject; (b)
amelioration of a disease or medical condition, e.g. elimination or regression of the disease or medical condition in a patient; (c) inhibition of the disease or medical condition, e.g. (d) including alleviation of symptoms of the disease or medical condition in the patient;

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length. Unless otherwise indicated, "polypeptide", "peptide" and "protein" refer to genetically encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, as well as polypeptides with modified peptide backbones. The term includes, but is not limited to fusion proteins, fusion proteins containing heterologous amino acid sequences,
Fusions containing heterologous (heterologous) and homologous (homologous) leader sequences, proteins containing at least one N-terminal methionine residue (e.g., to facilitate production in recombinant bacterial host cells), immunological including proteins tagged to .

"Native amino acid sequence" or "parent amino acid sequence" are used interchangeably herein to refer to the amino acid sequence of a polypeptide prior to modification to contain modified amino acid residues.

The terms "amino acid analogue", "unnatural amino acid" and the like can be used interchangeably and refer to amino acids that are similar in structure and/or overall shape to one or more amino acids commonly found in naturally occurring proteins. containing amino acid-like compounds (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include naturally occurring amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs having the same stereochemistry as the naturally occurring D-forms, and L-forms of amino acid analogs. In some instances, the amino acid analogue is one or more natural amino acid backbones (
backbone) structure and/or side chain structure, the difference being one or more modifying groups within the molecule. Such modifications include, but are not limited to, substitution of an atom (e.g., N) by a related atom (e.g., S), a group (e.g., methyl or hydroxyl, etc.) or an atom (e.g.,
(eg, Cl or Br), deletion of groups, substitution of covalent bonds (such as replacement of single bonds by double bonds), or combinations thereof. For example, an amino acid analogue is α-
Hydroxy acids and α-amino acids and the like can be included.

Terms such as "amino acid side chain" or "side chain of an amino acid" can be used to denote a substituent attached to the α-carbon of an amino acid residue, including natural amino acids, unnatural amino acids and amino acid analogs. Amino acid side chains can also include the amino acid side chains described in the context of the modified amino acids and/or conjugates described herein.

Terms such as "carbohydrate" may be used to denote monomeric units and/or polymers of monosaccharides, disaccharides, oligosaccharides and polysaccharides. The term sugar may be used to denote smaller carbohydrates such as monosaccharides, disaccharides. The term "carbohydrate derivative" means that one or more of the functional groups of the carbohydrate of interest have been substituted (by any convenient substituent) or modified (using any convenient chemical method). converted to another group) or absent (e.g. removed or substituted by H). A variety of carbohydrates and carbohydrate derivatives are available and can be adapted for use in the subject compounds and conjugates.

The term "antibody" is used in its broadest sense and includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (e.g. bispecific antibodies).
, humanized antibodies, single chain antibodies, chimeric antibodies, antibody fragments (eg, Fab fragments), and the like. Antibodies can bind to target antigens (Janeway, C., Travers
, P. , Walport, M.; , Shlomchik (2001) Immuno Bio
Logic, 5th Ed. , Garland Publishing, New York
). A target antigen may have one or more binding sites, also called epitopes, recognized by the complementarity determining regions (CDRs) formed by one or more variable regions of an antibody.

The term "native antibody" refers to an antibody in which the heavy and light chains of the antibody are produced and paired by the immune system of a multicellular organism. Spleen, lymph nodes, bone marrow and serum are examples of tissues that produce natural antibodies. For example, antibodies produced by antibody-producing cells isolated from the original animal immunized with the antigen are natural antibodies.

The terms "humanized antibody" or "humanized immunoglobulin" refer to non-human (e.g., framework regions, constant regions or CDRs) that contain one or more amino acids (e.g., framework regions, constant regions or CDRs) substituted with amino acids at corresponding positions from a human antibody. eg mouse or rabbit) antibodies. Generally, humanized antibodies generate a reduced immune response in a human host compared to non-humanized forms of the same antibody. Antibodies can be humanized using various techniques known in the art, such techniques including, for example, CDR grafting (EP 239,400; PC
T Publication WO 91/09967; U.S. Patent Nos. 5,225,539, 5,530,10
1 and 5,585,089), veneering or resurfacing (EP 5
92, 106; EP 519, 596; Padlan, Molecular Immun
28(4/5):489-498 (1991);
Protein Engineering 7(6):805-814 (1994);
guska et al., PNAS 91:969-973 (1994)), and strand shuffling (US Pat. No. 5,565,332). In certain embodiments, modeling of CDR and framework residue interactions to identify framework residues important for antigen binding and sequence comparison to identify aberrant framework residues at specific positions to identify framework substitutions (e.g., US Pat. No. 5,585,08
9; Riechmann et al., Nature 332:323 (1988)). Additional methods for humanizing antibodies contemplated for use in the present invention are disclosed in U.S. Pat.
70,403, 5,698,417, 5,693,493, 5,558,8
64, 4,935,496 and 4,816,567 and PCT Publication WO
98/45331 and WO 98/45332. In certain embodiments, the subject rabbit antibodies are US20040086979 and US20050033.
031. Thus, said antibodies can be humanized using methods well known in the art.

The term "chimeric antibody" is derived from antibody variable and constant region genes belonging to different species,
Typically, it means an antibody in which light chain and heavy chain genes have been constructed by genetic engineering. For example, the variable segments of genes derived from murine monoclonal antibodies are gamma1 and gamma3.
can be linked to human constant segments such as An example of a therapeutic chimeric antibody is a hybrid protein composed of a variable or antigen binding domain derived from a murine antibody and a constant or effector domain derived from a human antibody, although domains from other mammalian species can also be used.

An immunoglobulin polypeptide immunoglobulin light or heavy chain variable region consists of a framework region (FR) interspersed with three hypervariable regions (also called "complementarity determining regions" or "CDRs"). be done. Framework regions and CDR ranges have been determined ("Sequences of Proteins of Immunological
al Interest", E. Kabat et al., U.S. Department of
See Health and Human Services, 1991). The framework regions of antibodies are the combined framework regions of the constituent light and heavy chains, CD
It serves to position and align R. CDRs are primarily responsible for binding epitopes of the antigen.

Throughout this disclosure, the numbering of residues in immunoglobulin heavy chains and immunoglobulin light chains is according to Kabat et al., Sequences of Proteins of Im
Munological Interest, 5th Ed. public health
h Services, National Institutes of Health,
Bethesda, Md. (1991), which is expressly incorporated herein by reference.

A "parental Ig polypeptide" is a polypeptide comprising an amino acid sequence lacking an aldehyde-tagged constant region as described herein. A parent polypeptide can include a native sequence constant region or can include constant regions containing pre-existing amino acid sequence modifications (eg, additions, deletions and/or substitutions).

In the context of Ig polypeptides, the term "constant region" is well understood in the art and refers to the C-terminal region of an Ig heavy or Ig light chain. The Ig heavy chain constant region consists of CH1, CH2 and CH3 domains (and CH if the heavy chain is a mu or epsilon heavy chain).
4 domains). In native Ig heavy chains, the CH1, CH2, CH3 (and CH4 if present) domains begin immediately (C-terminal) after the heavy chain variable (VH) region and are each about 100 to about 130 amino acids in length. have In native Ig light chains, the constant region begins immediately (C-terminal) after the light chain variable (VL) region and is approximately 100 to 120 amino acids long.
It has a length of amino acids.

As used herein, the term "CDR" or "complementarity determining region" is intended to mean the discrete antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described in Kabat et al., J. Am. Biol. Chem. 252: 6609-6616 (19
77); Kabat et al. S. Dept. of Health and Human
Services, "Sequences of proteins of immunity
logical interest"(1991); Chothia et al., J. Mol.
Biol. 196:901-917 (1987); and MacCallum et al., J. Am. M.
ol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, the application of any definition to refer to the CDRs of an antibody or grafted antibody or variant thereof is intended to be within the terms defined and used herein. The amino acid residues comprising the CDRs defined by each of the above references are shown in Table 1 below for comparison.

"Genetically encodable" as used in reference to an amino acid sequence of a polypeptide, peptide or protein means that said amino acid sequence is composed of amino acid residues that can be produced by transcription and translation of the nucleic acid encoding said amino acid sequence. , wherein transcription and/or translation can occur in a cell or in a cell-free in vitro transcription/translation system.

The term "control sequence" refers to a DNA sequence that facilitates the expression of an operably linked coding sequence in a particular expression system (eg, mammalian cells, bacterial cells, cell-free synthesis, etc.).
Suitable regulatory sequences for prokaryotic systems include, for example, promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic systems may utilize promoters, polyadenylation signals and enhancers.

A nucleic acid is said to be "operably linked" when it is placed into a functional relationship to another nucleic acid sequence. For example, the DNA of a pre-sequence or secretory leader is said to be operably linked to the DNA of a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide, the promoter or An enhancer is said to be operably linked to a coding sequence if it affects the transcription of that sequence; or a ribosome binding site is said to be operably linked to a coding sequence. , if it is positioned so as to facilitate initiation of translation. in general,
"Operatively linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. . Linking is accomplished by a ligation or amplification reaction. Synthetic oligonucleotide adapters or linkers can be used to join sequences, in accordance with common practice.

The term "expression cassette" as used herein refers to, for example, the use of restriction sites suitable for ligation into a construct of interest or homologous recombination into the construct of interest or into the host cell genome. by) means a segment of a nucleic acid (usually DNA) that can be inserted into a nucleic acid. Generally, the nucleic acid segment comprises a polynucleotide encoding a polypeptide of interest, and the cassette and restriction sites are designed to facilitate insertion of the cassette in the proper reading frame for transcription and translation. An expression cassette can also contain elements that facilitate expression of a polynucleotide encoding a polypeptide of interest in a host cell. These elements include, but are not limited to, promoters, minimal promoters, enhancers, response elements, terminator sequences, polyadenylation sequences and the like.

As used herein, the term "isolated" is intended to indicate that the compound of interest is present in an environment different from that in which the compound naturally occurs. "Isolated"
"" is meant to include that the compound is present in a sample that is substantially enriched for the compound of interest and/or that the compound of interest is partially or substantially purified.

As used herein, the term "substantially purified" means that a compound has been substantially removed from its natural environment and is at least 60% free from other components that naturally accompany it.
, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 98% or more free.

The term "physiological conditions" refers to conditions compatible with living cells, e.g. temperature compatible with living cells, p
It is intended to include predominantly aqueous conditions such as H, salinity, and the like.

"Reaction partner" means a molecule or portion of a molecule that specifically reacts with another reaction partner to produce a reaction product. Typical reaction partners include the sulfatase motif and the cysteine or serine of formylglycine-generating enzyme (FGE), which converts aldehyde tags containing formylglycine (FGly) instead of cysteine or serine in the motif. React to produce reaction products. Other typical reaction partners include the aldehyde (e.g., reactive aldehyde group) of the fGly residue of the converted aldehyde tag and an "aldehyde-reactive reaction partner," which is an aldehyde-reactive group and a moiety of interest. to produce a reaction product of a modified aldehyde-tagged polypeptide having a moiety of interest conjugated to the modified polypeptide via a modified gGly residue.

"N-terminus" means the terminal amino acid residue of a polypeptide having a free amine group,
Amine groups at terminal amino acid residues usually form part of the covalent backbone of a polypeptide.

"C-terminus" refers to the terminal amino acid residue of a polypeptide having a free carboxyl group, the carboxyl groups at non-C-terminal amino acid residues usually forming part of the covalent backbone of the polypeptide.

An "internal site" as used in reference to a polypeptide or amino acid sequence of a polypeptide means a region of the polypeptide that is not present at the N-terminus or C-terminus.

Before describing the invention in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. Also, since the terminology used herein is for the purpose of describing particular embodiments only, and the scope of the invention is limited only by the appended claims, the terminology should be understood as non-limiting.

When a range of values is stated, each intervening value between the upper and lower limits of the range up to tenths of the unit of the lower limit, unless the context clearly contradicts, and within the stated range Any other indicated or intervening value of is understood to be included in the invention. It is understood that the upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and that any specifically excluded limit in the stated range may be present. As a premise,
It is included in the present invention as well. Where the stated range includes one or both of those limits, ranges excluding either or both of those included limits are also included in the invention.

Although certain features of the invention are described in the context of separate embodiments for clarity, it is understood that they can also be provided in combination in a single embodiment. Conversely, various features of the invention, although for brevity have been described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. is. All combinations of embodiments pertaining to the present invention are subject matter wherein such combinations are compounds that are, for example, stable compounds (i.e., compounds that can be prepared, isolated, characterized, and tested for biological activity). To the extent included, each individual combination is specifically included in the present invention and disclosed herein, as if each individual combination were individually and expressly disclosed herein. again,
All subcombinations of various embodiments and elements thereof (e.g., elements of chemical groups recited in embodiments describing such variations) also include each individual subcombination individually and expressly. specifically included in the present invention and disclosed herein, as if explicitly disclosed herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It should be noted that the singular forms as used in this specification and the appended claims include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional (ie, included if desired) element. Accordingly, this statement is "exclusively" with respect to the recitation of claim elements,
It is intended to serve as an antecedent basis for the use of exclusive terms such as "only" or the use of "negative" limitations.

Although certain features of the invention are described in the context of separate embodiments for clarity, it is understood that they can also be provided in combination in a single embodiment. Conversely, various features of the invention, although for brevity have been described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. is.

The publications mentioned herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication as prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently verified.

DETAILED DESCRIPTION The present disclosure provides anti-CD22 antibody-maytansine conjugate constructs. The disclosure also includes methods of making such conjugates and methods of using them. Each embodiment is described in further detail in the following sections.

Antibody-Drug Conjugates The present disclosure provides conjugates, eg, antibody-drug conjugates. "Conjugate" means a first moiety (eg, antibody) stably attached to a second moiety (eg, drug). For example, a maytansine conjugate comprises a maytansine (eg, a maytansine active agent moiety) stably attached to another moiety (eg, an antibody). "Stably attached" means that a moiety is bound to another moiety or structure under standard conditions. In some embodiments, the first portion and the second portion are attached to each other by one or more covalent bonds.

In certain embodiments, the conjugate is a polypeptide conjugate comprising a polypeptide conjugated to a second moiety. In certain embodiments, the moiety conjugated to the polypeptide is any of a variety of moieties of interest, such as detectable labels, drugs, water-soluble polymers, or immobilization of the polypeptide to membranes or surfaces. It can be a part (but not limited to) for conversion. In some embodiments, the conjugate is a maytansine conjugate, wherein the polypeptide is conjugated to a maytansine or a maytansine active agent moiety. "Maytansine", "maytansine portion", "maytansine active substance portion" and "maytansinoid" mean maytansine and analogs and derivatives thereof, and pharmaceutically active maytansine portions and/or portions thereof. The maytansine that is conjugated to the polypeptide can be any of a variety of maytansinoid moieties, including, but not limited to, maytansines and analogs and derivatives thereof as described herein.
can be

The moiety of interest can be conjugated to the polypeptide at any desired site on the polypeptide. Accordingly, the present disclosure provides modified polypeptides having moieties conjugated, eg, at or near the C-terminus of the polypeptide. Other examples include modified polypeptides having moieties conjugated at or near the N-terminus of the polypeptide. Specific examples also include modified polypeptides having moieties conjugated at a position between the C-terminus and N-terminus of the polypeptide (eg, at an internal site of the polypeptide). Combinations of the foregoing are also possible when the modified polypeptide is conjugated to more than one moiety.

In certain embodiments, conjugates of the present disclosure comprise maytansine conjugated to an amino acid residue of a polypeptide at the α-carbon of the amino acid residue. In other words, the maytansine conjugate is such that the side chains of one or more amino acid residues in the polypeptide are attached to maytansine (e.g., attached to maytansine via a linker described herein). (b) modified, including polypeptides. For example, a maytansine conjugate is such that the α-carbon of one or more amino acid residues in the polypeptide is
Included are polypeptides that have been modified to bind to maytansine (eg, to bind to maytansine via a linker described herein).

Embodiments of the present disclosure include one or more sections, such as 2 sections, 3 sections, 4 sections, 5 sections.
Conjugates in which the polypeptide is conjugated to 1, 6, 7, 8, 9, or 10 or more moieties. The moiety can be conjugated to the polypeptide at one or more sites on the polypeptide. For example, one or more moieties can be conjugated to a single amino acid residue of the polypeptide. In some cases, one moiety is conjugated to an amino acid residue of the polypeptide. In other embodiments, two moieties may be conjugated to the same amino acid residue of the polypeptide. In other embodiments, the first portion is conjugated to the first amino acid residue of the polypeptide and the second portion is conjugated to the second amino acid residue of the polypeptide.
Combinations of the foregoing are also possible, eg the polypeptide is conjugated to a first moiety at the first amino acid residue and to two other moieties at the second amino acid residue. Other combinations are also possible, for example and without limitation, the polypeptide is conjugated to the first and second moieties at the first amino acid residue and the third and fourth moieties at the second amino acid residue. conjugated to, and so on.

The one or more polypeptide amino acid residues conjugated to one or more moieties can be naturally occurring amino acids, non-natural amino acids, or combinations thereof. For example, a conjugate can include a portion of a polypeptide conjugated to naturally occurring amino acid residues. In other examples, a conjugate can include a moiety conjugated to a non-natural amino acid residue of a polypeptide. One or more moieties can be conjugated to the polypeptide at said single natural or unnatural amino acid residue. One or more natural or non-natural amino acid residues in a polypeptide can be conjugated to one or more moieties described herein. For example, two (or more) amino acid residues (e.g., natural or non-natural amino acid residues) in a polypeptide are
Multiple sites in a polypeptide can be modified by conjugation to individual moieties.

As described herein, polypeptides can be conjugated to one or more moieties. In some embodiments, the moiety of interest is a chemical moiety such as a drug or detectable label. For example, a drug (eg, maytansine) can be conjugated to the polypeptide, or, in other embodiments, a detectable label can be conjugated to the polypeptide. Thus, for example, embodiments of the present disclosure include, but are not limited to: a conjugate of a polypeptide and a drug, a conjugate of a polypeptide and a detectable label, two or more drugs and Conjugates with polypeptides, conjugates of two or more detectable labels with a polypeptide, and the like.

In some embodiments, the polypeptide and moiety of interest are conjugated via a coupling moiety. For example, the polypeptide and portion of interest can each be
It is possible to bind (e.g., covalently) to the coupling moiety, thus indirectly binding the polypeptide and the moiety of interest (e.g., a drug such as maytansine) together through the coupling moiety. is possible. In some cases, the coupling moiety comprises a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound, or a derivative of a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound. For example, a general scheme for attaching a moiety of interest (eg, maytansine) to a polypeptide via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety is shown in the general reaction scheme below. . Hydrazinyl-indolyl and hydrazinyl-pyrrolo-pyridinyl coupling moieties are
As used herein, hydrazino-iso-pictet-Spengler (HIP
Also referred to as S) coupling moieties and aza-hydrazino-iso-pictet-Spengler (aza-HIPS) coupling moieties.

In the above reaction scheme, R is the moiety of interest (eg, maytansine) that is conjugated to the polypeptide. As shown in the reaction scheme above, a polypeptide containing a 2-formylglycine residue (fGly) is transformed to include a coupling moiety (eg, a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety). Reaction with a modified drug (eg, maytansine) yields a Polypeptide Conjugate attached to a coupling moiety, thus attaching maytansine to the polypeptide via the coupling moiety.

As described herein, the moiety can be any of a variety of moieties including, but not limited to, chemical moieties such as detectable labels or drugs (e.g., maytansinoids). and R′ and R″ are each independently any desired substituent such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl , substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl , substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. Z can be CR ¹¹ , NR ¹² , N, O or S, where R ¹¹ and R ¹² are each independently substituted as described for R′ and R″ above. is selected from any of the groups.

As shown in the conjugates and compounds described herein,
Other hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moieties are also possible. For example, a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety can be modified to attach (eg, covalently attach) to a linker. Accordingly, embodiments of the present disclosure provide a drug (e.g., maytansine) via a linker.
including hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moieties attached to. Various embodiments of linkers that can attach hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moieties to drugs (eg, maytansine) are described in detail herein.

In certain embodiments, the polypeptide may be modified prior to conjugation to the moiety of interest and the polypeptide to the moiety of interest. Modification of a polypeptide can provide a modified polypeptide containing one or more reactive groups suitable for conjugation to a moiety of interest. In some cases, the polypeptide is modified at one or more amino acid residues to create a moiety of interest (e.g., a coupling moiety such as the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moieties described above). part including
can provide one or more reactive groups suitable for bonding to For example, a polypeptide can be modified to contain a reactive aldehyde group (eg, a reactive aldehyde). A reactive aldehyde can be included in an "aldehyde tag" or "ald-tag," which terms, as used herein, are 2-formylglycine residues (herein referred to as " F.
An amino acid sequence derived from a sulfatase motif (eg, L(C/S)TPSR) that has been converted by the action of formylglycine-generating enzyme (FGE) to contain a sulfatase motif (referred to as "Gly"). FGly residues produced by FGE may also be referred to as "formylglycine." In other words, the term "aldehyde tag" includes a "converted" sulfatase motif (i.e., a sulfatase motif in which a cysteine or serine residue has been converted to FGly by the action of FGE, such as L(FGly)TPSR). Used herein to indicate an amino acid sequence. A sulfatase motif that is converted is an "unconverted" sulfatase motif (i.e., a sulfatase motif in which a cysteine or serine residue has not yet been converted to FGly by the action of FGE, but is convertible, e.g., sequence L(C/ S) an unconverted sulfatase motif with TPSR). formylglycine synthase (FG) to the sulfatase motif
"Conversion" used in the context of the action of E) is the biochemical modification of a cysteine or serine residue in the sulfatase motif to a formylglycine (FGly) residue (e.g. C
ys to FGly or Ser to FGly). Additional embodiments of aldehyde tags and their use in site-specific protein modification are described in US Pat. No. 7,985,
783 and US Pat. No. 8,729,232, the disclosures of each of which are incorporated herein by reference.

In some cases, modified polypeptides containing an FGly residue can be treated with a compound of interest by reaction of FGly with a compound (eg, a compound containing a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety). It can be conjugated to a moiety. For example, an FGly-containing polypeptide can be contacted with a drug containing reaction partner under conditions suitable to effect conjugation of the drug to the polypeptide. In some cases, drug containing reaction partners may include hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moieties described above. For example, maytansine may be modified to contain a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety. In some cases, maytansine is attached to hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl, for example, via a linker to hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl, as described in detail herein. Covalently bond.

In certain embodiments, a conjugate of the disclosure comprises a polypeptide (eg, an antibody, eg, an anti-CD22 antibody) with at least one modified amino acid residue. Modified amino acid residues of the polypeptides are hydrazinyl-indolyl or hydrazinyl-
It can be coupled (conjugated) to drugs containing a pyrrolo-pyridinyl coupling moiety (eg, maytansine). In some embodiments, polypeptides (e.g., anti-CD22
Antibodies) modified amino acid residues can be derived from cysteine or serine residues which are converted to FGly residues as described above. In certain embodiments, a drug containing a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety described above is added with FGl.
The y residue is conjugated to give conjugates of the disclosure, where the drug is conjugated to the polypeptide via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety. The term FGly' as used herein is
It refers to modified amino acid residues of a polypeptide (eg, an anti-CD22 antibody) that is coupled to a moiety of interest (eg, a drug such as a maytansinoid).

In certain embodiments, the conjugate comprises at least one modified amino acid residue of Formula (I) as described herein. For example, the conjugate can comprise at least one modified amino acid residue with a side chain of formula (I).

[In the formula,
Z is ^CR4 or N;
R ¹ is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
selected from substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl;
R ² and R ³ are each independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acylamino, selected from aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, or ^R2 and R ³ may optionally be cyclically linked to form a 5- or 6-membered heterocyclyl;
Each ^R4 is independently hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acylamino,
selected from aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl;
L is −(T ¹ −V ¹ ) _a −(T ² −V ² ) _b −(T ³ −V ³ ) _c −(T ⁴ −V ⁴ )
_d -comprising linkers, where a, b, c and d are each independently 0 or 1 and the sum of a, b, c and d is 1-4;
T ¹ , T ² , T ³ and T ⁴ are each independently (C ₁ -C ₁₂ )alkyl, substituted (C ₁ -C ₁₂ )alkyl, (EDA) _w , (PEG) _n , (AA) _p , −(CR ¹³
OH) selected from _h- , piperidine-4-amino (4AP), acetal groups, hydrazines, disulfides and esters, where EDA is an ethylenediamine moiety, PE
G is polyethylene glycol or modified polyethylene glycol, AA is an amino acid residue, w is an integer from 1 to 20, n is an integer from 1 to 30, p is an integer from 1 to 20, h is an integer from 1 to 12;
V ¹ , V ² , V ³ and V ⁴ are each independently a covalent bond, -CO-, -NR ^1
5 -, -NR ¹⁵ (CH ₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -N
R ¹⁵ CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —S
selected from the group consisting of O ₂ —, —SO ₂ NR ¹⁵ —, —NR ¹⁵ SO ₂ — and —P(O)OH—, wherein q is an integer from 1 to 6;
each R ¹³ is independently selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl;
Each R ¹⁵ is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cyclo selected from alkyl, heterocyclyl and substituted heterocyclyl;
W1 is ^a maytansinoid; and W2 is an anti ^- CD22 antibody].

In some embodiments, Z is ^CR4 or N. ^In some embodiments, Z is CR4. In some embodiments, Z is N.

In some embodiments, R ¹ is from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. selected. In some embodiments, R ¹ is hydrogen. In certain embodiments, R ¹ is alkyl or substituted alkyl, such as C _1-6 alkyl or C
_1-6 substituted alkyl, alternatively C _1-4 alkyl or C _1-4 substituted alkyl, alternatively C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ¹ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3
alkenyl or C _2-3 substituted alkenyl; In certain embodiments, R ¹ is alkynyl or substituted alkynyl, for example C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ¹ is aryl or substituted aryl, eg, C _5-8 aryl or C _5-8 substituted aryl, eg,
_C5 aryl or _C5 substituted aryl, or _C6 aryl or _C6 substituted aryl. In certain embodiments, R ¹ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl aryl or C6 _- substituted heteroaryl; In certain embodiments, R ¹ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, or C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In some embodiments, R ¹ is heterocyclyl or substituted heterocyclyl, eg, C _3-8 heterocyclyl or C _3-8
substituted heterocyclyl, alternatively C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, alternatively C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In some embodiments, R ² and R ³ are each independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,
substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl,
selected from acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, or R ² and R ³ may optionally be cyclically linked to form a 5- or 6-membered heterocyclyl.

In some embodiments, ^R2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acylamino, aminoacyl, alkyl amido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. In some embodiments, ^R2 is hydrogen. In certain embodiments, R ² is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ² is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-
4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-
It is ₃ -substituted alkenyl. In some embodiments, ^R2 is alkynyl or substituted alkynyl. In some embodiments, ^R2 is alkoxy or substituted alkoxy.
In some embodiments, ^R2 is amino or substituted amino. In some embodiments, ^R2 is carboxyl or carboxylester. In some embodiments,
^R2 is acyl or acyloxy. In some embodiments, ^R2 is acylamino or aminoacyl. In some embodiments, R ² is alkylamido or substituted alkylamido. In some embodiments, ^R2 is sulfonyl. In some embodiments, ^R2 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ² is aryl or substituted aryl, for example C _5-8 aryl or C _{5-
8 -} substituted aryl, such as C5 aryl or C5 _- substituted aryl, or C6 aryl or _{C6 -} substituted aryl. In certain embodiments, R ² is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl aryl or C6 _- substituted heteroaryl; In certain embodiments, R ² is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, or C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl,
Alternatively, it is C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In some embodiments, R ² is heterocyclyl or substituted heterocyclyl, eg, C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

^In some embodiments, R3 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acylamino, aminoacyl, alkyl amido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. ^In some embodiments, R3 is hydrogen. In certain embodiments, R ³ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ³ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-
4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-
It is ₃ -substituted alkenyl. ^In some embodiments, R3 is alkynyl or substituted alkynyl. ^In some embodiments, R3 is alkoxy or substituted alkoxy.
^In some embodiments, R3 is amino or substituted amino. ^In some embodiments, R3 is carboxyl or carboxylester. In some embodiments,
^R3 is acyl or acyloxy. ^In some embodiments, R3 is acylamino or aminoacyl. In some embodiments, R ³ is alkylamido or substituted alkylamido. ^In some embodiments, R3 is sulfonyl. ^In some embodiments, R3 is thioalkoxy or substituted thioalkoxy. In some embodiments, R ³ is aryl or substituted aryl, for example C _5-8 aryl or C _{5-
8 -} substituted aryl, such as C5 aryl or C5 _- substituted aryl, or C6 aryl or _{C6 -} substituted aryl. In certain embodiments, R ³ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl aryl or C6 _- substituted heteroaryl; In certain embodiments, R ³ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, or C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl,
Alternatively, it is C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In some embodiments, R ³ is heterocyclyl or substituted heterocyclyl, for example C _3-8 heterocyclyl or C _3-8 substituted heterocyclyl, C _3-6 heterocyclyl or C _3-6
substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, R ² and R ³ are optionally cyclically linked to form a 5- or 6-membered heterocyclyl. In some embodiments, R ² and R ³ are cyclically linked to form a 5- or 6-membered heterocyclyl. In some embodiments, R ² and R ³ are cyclically linked to form a 5-membered heterocyclyl. In some embodiments, R ² and R ³ are cyclically linked to form a 6-membered heterocyclyl.

In some embodiments, each ^R4 is independently hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl , acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,
selected from substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl;

Various possibilities for each ^R4 are described in more detail below. In some embodiments, ^R4 is hydrogen. In some embodiments, each ^R4 is hydrogen. In some embodiments,
^R4 is halogen, eg F, Cl, Br or I; In some embodiments, R ⁴
is F. In some embodiments, ^R4 is Cl. In some embodiments, R
⁴ is Br. In some embodiments, ^R4 is I. In some embodiments,
R ⁴ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl is. In certain embodiments, R ⁴ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In some embodiments, ^R4 is alkynyl or substituted alkynyl. In some embodiments, ^R4 is alkoxy or substituted alkoxy. In some embodiments, ^R4 is amino or substituted amino. In some embodiments, ^R4 is carboxyl or carboxylester. In some embodiments, ^R4 is acyl or acyloxy. In some embodiments, ^R4 is acylamino or aminoacyl. In some embodiments, R ⁴ is alkylamido or substituted alkylamido. In some embodiments, ^R4 is sulfonyl. In some embodiments, ^R4 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ⁴ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆
Aryl or C6 _- substituted aryl (eg, phenyl or substituted phenyl). In certain embodiments, R ⁴ is heteroaryl or substituted heteroaryl, eg, C _5-
8 heteroaryl or C _5-8 substituted heteroaryl, for example C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ⁴ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, or C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ⁴ is heterocyclyl or substituted heterocyclyl, such as C _3-8 heterocyclyl or C _3-8 substituted heterocyclyl, C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-
5 heterocyclyl or C _3-5 substituted heterocyclyl.

In some embodiments, W1 is ^a maytansinoid. Further description of maytansinoids is found in the disclosure herein.

In some embodiments, W2 is an anti ^- CD22 antibody. Further description of anti-CD22 antibodies is found in the disclosure herein.

In certain embodiments, compounds of Formula (I) include a linker L. Linkers can be used to join the coupling moiety to one or more moieties of interest and/or one or more polypeptides. In some embodiments, a linker joins a coupling moiety to a polypeptide or chemical moiety. The linker may be attached (eg, covalently attached) to the coupling moiety at any convenient position. For example, a linker can attach a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety to a drug (eg, maytansine). A hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety can be used to conjugate a linker (and thereby a drug such as maytansine) to a polypeptide, such as an anti-CD22 antibody.

^In some embodiments, L links the coupling moiety to W1, thus the coupling moiety is indirectly linked to ^W1 through the linker L. As noted above, W1 is ^a maytansinoid, and thus L links the coupling moiety to the maytansinoid, eg, the coupling moiety indirectly links to the maytansinoid via a linker L.

Any convenient linker can be used in the present conjugates and compounds. In some embodiments, L is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acylamino, alkylamido, substituted alkylamido,
including groups selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. In some embodiments, L comprises an alkyl or substituted alkyl group. In some embodiments, L comprises an alkenyl or substituted alkenyl group. In some embodiments, L includes alkynyl or substituted alkynyl. In some embodiments, L comprises an alkoxy or substituted alkoxy group. In some embodiments, L comprises an amino or substituted amino group. In some embodiments, L comprises a carboxyl or carboxylester group.
In some embodiments, L contains an acylamino group. In some embodiments, L comprises an alkylamido or substituted alkylamido group. In some embodiments, L comprises an aryl or substituted aryl group. In some embodiments, L comprises a heteroaryl or substituted heteroaryl group. In some embodiments, L comprises a cycloalkyl or substituted cycloalkyl group. In some embodiments, L comprises a heterocyclyl or substituted heterocyclyl group.

In some embodiments, L comprises a polymer (polymer). For example, polymers include polyalkylene glycols and derivatives thereof such as polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol and propylene glycol (e.g., the homopolymers and copolymers may be substituted). or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinyl pyrrolidone, combinations thereof, and the like. In some embodiments, the polymer is polyalkylene glycol. In some embodiments, the polymer is polyethylene glycol. Other linkers are also possible, as demonstrated in the conjugates and compounds described in more detail below.

In some embodiments, L is of the formula -(L ¹ ) _a -(L ² ) _b -(L ³ ) _c -(L
⁴ ) A linker denoted by _d- , where L ¹ , L ² , L ³ and L ⁴ are each independently a linker unit and a, b, c and d are each independently , 0 or 1, and the sum of a, b, c and d is 1-4.

In some embodiments, the sum of a, b, c and d is one. In some embodiments, the sum of a, b, c and d is two. In some embodiments, the sum of a, b, c and d is three. In some embodiments, the sum of a, b, c and d is four. In some embodiments, a, b, c and d are each one. In some embodiments, a, b and c are each 1 and d is 0. In some embodiments,
a and b are each 1 and c and d are each 0; In some embodiments, a is 1 and b, c and d are each 0.

In certain embodiments, L ¹ is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety (eg, those shown in formula (I) above). ^In some embodiments, L2, if present, is attached to ^W1 .
^In some embodiments, L3 ^, if present, is attached to W1. ^In some embodiments, L4, if present, is attached to ^W1 .

Any convenient linker unit can be used in the linker. Linker units of interest include, but are not limited to, polymers such as polyethylene glycol, polyethylene and polyacrylate units, amino acid residues, carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, Included are aryl groups, heterocyclic groups, combinations thereof, and substituted forms thereof. In some embodiments, L ¹
, L ² , L ³ and L ⁴ (when present) each include polyethylene glycols, modified polyethylene glycols, amino acid residues, alkyl groups, substituted alkyls, aryl groups, substituted aryl groups and diamines (e.g., alkylenediamines). linking group).

In some embodiments, L ¹ (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residue, alkyl group, substituted alkyl, aryl group, substituted aryl group or diamine. ^In some embodiments, L1 comprises polyethylene glycol. ^In some embodiments, L1 comprises modified polyethylene glycol. In some embodiments, L1 comprises ^an amino acid residue. In some embodiments, L ¹ comprises an alkyl group or substituted alkyl. In some embodiments, L ¹ comprises an aryl group or substituted aryl group. In some embodiments, L ¹ comprises a diamine (eg, a linking group comprising an alkylenediamine).

In some embodiments, L ² (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residue, alkyl group, substituted alkyl, aryl group, substituted aryl group or diamine. In some embodiments, L2 ^comprises polyethylene glycol. In some embodiments, L2 ^comprises modified polyethylene glycol. In some embodiments, L2 ^comprises an amino acid residue. In some embodiments, L2 ^comprises an alkyl group or substituted alkyl. In some embodiments, L2 ^comprises an aryl group or substituted aryl group. In some embodiments, L2 ^comprises a diamine (eg, a linking group comprising an alkylenediamine).

In some embodiments, L ³ (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residue, alkyl group, substituted alkyl, aryl group, substituted aryl group or diamine. ^In some embodiments, L3 comprises polyethylene glycol. ^In some embodiments, L3 comprises modified polyethylene glycol. ^In some embodiments, L3 comprises an amino acid residue. ^In some embodiments, L3 comprises an alkyl group or substituted alkyl. ^In some embodiments, L3 comprises an aryl group or substituted aryl group. In some embodiments, L3 comprises a diamine ⁽ eg, a linking group comprising an alkylenediamine).

In some embodiments, L ⁴ (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residue, alkyl group, substituted alkyl, aryl group, substituted aryl group or diamine. ^In some embodiments, L4 comprises polyethylene glycol. ^In some embodiments, L4 comprises modified polyethylene glycol. ^In some embodiments, L4 comprises an amino acid residue. ^In some embodiments, L4 comprises an alkyl group or substituted alkyl. ^In some embodiments, L4 comprises an aryl group or substituted aryl group. ^In some embodiments, L4 comprises a diamine (eg, a linking group comprising an alkylenediamine).

In some embodiments, L is -(L ¹ ) _a -(L ² ) _b -(L ³ ) _c -(L ⁴
) _d is a linker containing -, where
-(L ¹ ) _a - is -(T ¹ -V ¹ ) _a -;
-(L ² ) _b - is -(T ² -V ² ) _b -;
-(L ³ ) _c - is -(T ³ -V ³ ) _c -; and -(L ⁴ ) _d - is -(T ⁴ -V ⁴ ) _d -;
where T ¹ , T ² , T ³ and T ⁴ , if present, are the linking strand (tether (te
ther)) group;
V ¹ , V ² , V ³ and V ⁴ , if present, are covalent bonds or linking functional groups; and a, b, c and d are each independently 0 or 1, a , b, c and d
is 1-4.

As noted above, in certain embodiments, L ¹ is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety (eg, those shown in formula (I) above). Thus, in certain embodiments, T ¹ is a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety (eg, the formula (
I) shown in). In some embodiments, V ¹ is attached to W ¹ (maytansinoid). ^In some embodiments, L2, when present, is
It binds to ^W1 . Thus, in certain embodiments, ^T2 , if present, is bound to ^W1 , or ^V2 , if present, is bound to ^W1 . ^In some embodiments, L3 ^, if present, is attached to W1. Thus, in certain embodiments, T3 ^, if present, is bound to W1 ^, or ^V3
is attached to ^W1 , if present. ^In some embodiments, L4, if present, is attached to ^W1 . Thus, in certain embodiments, ^T4 , if present, is bound to ^W1 , or ^V4 , if present, is bound to ^W1 .

With respect to tethering groups T ¹ , T ² , T ³ and T ⁴ , any convenient tethering group can be used in the present linkers. In some embodiments, T ¹ , T ² , T ³ and T ⁴ are each (C ₁ -C ₁₂ )alkyl, substituted (C ₁ -C ₁₂ )alkyl, (EDA) _w , (
PEG) _n , (AA) _p , —(CR ¹³ OH) _h —, piperidine-4-amino (4AP)
1 independently selected from , acetal groups, disulfides, hydrazines and esters
wherein w is an integer from 1-20, n is an integer from 1-30, p is an integer from 1-20, and h is an integer from 1-12.

In some embodiments, the sum of a, b, c and d is 2 and T ¹ -V ¹ , T ² -
n is not 6 when V ² , T ³ -V ³ , or T ⁴ -V ⁴ is (PEG) _n -CO. For example, in some cases the linker has the structure:

(where n is not 6).

In some embodiments, the sum of a, b, c and d is 2 and T ¹ -V ¹ , T ² -
When V ² , T ³ -V ³ , or T ⁴ -V ⁴ are (C ₁ -C ₁₂ )alkyl-NR ¹⁵ , (C ₁ -C ₁₂ )alkyl is not C ₅ -alkyl. For example, in some cases the linker has the structure:

(where g is not 4).

In some embodiments, linking chain groups (eg, T ¹ , T ² , T ³ and/or T ⁴ )
includes (C ₁ -C ₁₂ )alkyl or substituted (C ₁ -C ₁₂ )alkyl. In some embodiments, (C ₁ -C ₁₂ )alkyl has 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms. , or a straight or branched alkyl group containing 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, (C ₁ -C ₁₂ )alkyl is alkyl or substituted alkyl, such as C ₁ -C ₁₂ alkyl, or C ₁ -C ₁₀ alkyl, or C ₁ .
—C ₆ alkyl, or C ₁ -C ₃ alkyl. In some cases (C
₁ -C ₁₂ )alkyl is C ₂ -alkyl. For example, (C ₁ -C ₁₂ )alkyl can be alkylene or substituted alkylene, such as C ₁ -C ₁₂ alkylene, or C ₁ -C ₁₀ alkylene, or C ₁ -C ₆ alkylene, or C ₁ -C ₃ alkylene. . In some cases, (C ₁ -C ₁₂ )alkyl is C ₂ -alkylene.

In certain embodiments, substituted (C ₁ -C ₁₂ )alkyl has 1-12 carbon atoms,
for example 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms,
or a linear or branched substituted alkyl group containing 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, substituted (C ₁ -C ₁₂
) alkyl is substituted alkyl, such as substituted C ₁ -C ₁₂ alkyl, or substituted C ₁ -C ₁₀
It can be alkyl, or substituted C ₁ -C ₆ alkyl, or substituted C ₁ -C ₃ alkyl.
In some cases, substituted (C ₁ -C ₁₂ )alkyl is substituted C ₂ -alkyl.
For example, substituted (C ₁ -C ₁₂ )alkyl is substituted alkylene, such as substituted C ₁ -C ₁₂ alkylene, or substituted C ₁ -C ₁₀ alkylene, or substituted C ₁ -C ₆ alkylene, or substituted C ₁ -C ₃ alkylene. can be In some cases, substituted (C ₁ -C ₁₂ )
Alkyl is substituted C ₂ -alkylene.

In some embodiments, linking chain groups (eg, T ¹ , T ² , T ³ and/or T ⁴ )
contains ethylenediamine (EDA) moieties, eg, EDA-containing linkages. In some embodiments, (EDA) _w comprises one or more EDA moieties, where, for example, w is an integer from 1-50, such as an integer from 1-40, an integer from 1-30, an integer from 1-20, integer, integer from 1 to 12, 1
an integer from ˜6, such as 1, 2, 3, 4, 5 or 6. Linked ethylenediamine (ED
A) moieties may be optionally substituted at one or more convenient positions with any convenient substituent such as alkyl, substituted alkyl, acyl, substituted acyl, aryl or substituted aryl. In some embodiments, the EDA moiety has the structure:

wherein y is an integer from 1 to 6, r is 0 or 1, and each R ¹² is
independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, selected from substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl; In some embodiments, y is 1, 2, 3, 4, 5, or 6. In some embodiments, y is 1 and r is 0. In some embodiments, y is 1 and r is 1. In some embodiments, y is 2 and r is 0. In some embodiments, y is 2;
r is 1; In some embodiments, each R ¹² is independently selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl. In some embodiments, ED
Any two adjacent R ¹² groups of A may be cyclically linked to form, for example, a piperazinyl ring. In some embodiments, y is 1 and two adjacent R ¹² groups are alkyl groups and are cyclically linked to form a piperazinyl ring. In some embodiments, y is 1 and adjacent R ¹² groups are selected from hydrogen, alkyl (eg methyl) and substituted alkyl (eg lower alkyl-OH such as ethyl-OH or propyl-OH). be.

In some embodiments, the linking chain group comprises a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidine-4-amino, P4A). The 4AP part is
It can optionally be substituted at one or more convenient positions with any convenient substituent such as alkyl, substituted alkyl, polyethylene glycol moieties, acyl, substituted acyl, aryl or substituted aryl. In some embodiments, the 4AP moiety has the structure:

wherein R ¹² is hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety (e.g. polyethylene glycol or modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,
amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted selected from cycloalkyl, heterocyclyl and substituted heterocyclyl; In some embodiments, R ¹² is carboxy-modified polyethylene glycol.

In certain embodiments, R ¹² comprises a polyethylene glycol moiety represented by the formula (PEG) _k , which has the structure:

where k is 1 to 20, such as 1 to 18, or 1 to
16, or 1-14, or 1-12, or 1-10, or 1-8, or 1-6
, or an integer from 1 to 4, or 1 or 2, such as 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some cases, k is two. In some embodiments, R ¹⁷ is OH, C
is selected from OOH or COOR, wherein R is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl; is selected from In some embodiments, R ¹⁷ is COOH.

In some embodiments, linking chain groups (eg, T ¹ , T ² , T ³ and/or T ⁴ )
contains (PEG) _n , where (PEG) _n is a polyethylene glycol or modified polyethylene glycol linking unit. In some embodiments, (PEG) _n has the structure:

wherein n is 1-50, such as 1-40, 1-30, 1-20, 1-12
or an integer from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 or 20. In some cases n is two. In some cases n is three. In some cases n
is 6. In some cases, n is twelve.

In some embodiments, linking chain groups (eg, T ¹ , T ² , T ³ and/or T ⁴ )
contains (AA) _p , where AA is an amino acid residue. Any convenient amino acid can be used. Amino acids of interest include, but are not limited to, L- and D-amino acids, naturally occurring amino acids such as any of the 20 major alpha-amino acids and beta-alanine, unnatural amino acids such as , amino acid analogs), such as unnatural alpha-amino acids and/or unnatural beta-amino acids. In some embodiments, p is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12 or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1
5, 16, 17, 18, 19 or 20. In some embodiments, p is 1. In some embodiments, p is two.

In some embodiments, linking chain groups (eg, T ¹ , T ² , T ³ and/or T ⁴ )
contains a moiety represented by the formula: -(CR ¹³ OH) _h -, where h is 0, or
Alternatively n is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12 or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11 or 12. In some embodiments, h is 1. In some embodiments, h is two. In some embodiments, R ₁₃ is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acylamino, aminoacyl, alkyl amido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. In some embodiments, R ¹³ is hydrogen. In certain embodiments, R ¹³ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or _C1
-3 substituted alkyl. In certain embodiments, R ¹³ is alkenyl or substituted alkenyl, for example C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4
alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3
substituted alkenyl; In some embodiments, R ¹³ is alkynyl or substituted alkynyl. In some embodiments, R ¹³ is alkoxy or substituted alkoxy. In some embodiments, R ¹³ is amino or substituted amino. In some embodiments, R ¹³ is carboxyl or carboxyl ester. In some embodiments, R ¹³ is acyl or acyloxy. In some embodiments, R ¹³ is acylamino or aminoacyl. In some embodiments, R ¹³ is alkylamido or substituted alkylamido. In some embodiments, R ¹³ is sulfonyl. In some embodiments, R ¹³ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ¹³ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. be. In some embodiments, R ¹³ is heteroaryl or substituted heteroaryl, for example C _5-8 heteroaryl or C _5
-8 substituted heteroaryl, such as _C5 heteroaryl or _C5 substituted heteroaryl,
Or _C6 heteroaryl or _C6 substituted heteroaryl. In certain embodiments, R ¹³ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, or C _3-6 cycloalkyl or C _3-
6 -substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ¹³ is heterocyclyl or substituted heterocyclyl, such as C _3-8 heterocyclyl or C _3-8 substituted heterocyclyl, C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In some embodiments, R ¹³ is selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl. ^In these embodiments, alkyl, substituted alkyl, aryl and substituted aryl are as described above for R13.

With respect to linking functionalities V ¹ , V ² , V ³ and V ⁴ , any convenient linking functionalities can be used in the linker. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide,
Includes sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, thiophosphoridate, and the like. In some embodiments, V ¹ , V ² , V ³ and V ⁴ are each independently a covalent bond, —CO—, —NR ¹⁵ —, —NR ¹⁵ (CH
₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -NR ¹⁵ CO-, -C(O
) O—, —OC(O)—, —O—, —S—, —S(O)—, —SO ₂ —, —SO ₂ NR ^1
5 -, -NR ¹⁵ SO ₂ - and -P(O)OH-, wherein q
is an integer from 1 to 6. In some embodiments, q is an integer from 1 to 6 (eg, 1, 2,
3, 4, 5 or 6). In some embodiments, q is one. In some embodiments, q is two.

In some embodiments, each R ¹⁵ is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl , acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl,
selected from thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl.

Various possibilities for each ^R15 are described in more detail below. In some embodiments, R ^1
5 is hydrogen. In some embodiments, each R ¹⁵ is hydrogen. In certain embodiments, R ¹⁵ is alkyl or substituted alkyl, for example C _1-6 alkyl or C _1-6
substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-
3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ¹⁵ is alkenyl or substituted alkenyl, for example C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In some embodiments, R ¹⁵ is alkynyl or substituted alkynyl. In some embodiments, R ¹⁵ is alkoxy or substituted alkoxy. In some embodiments, R ¹⁵ is amino or substituted amino. In some embodiments, R ¹⁵ is carboxyl or carboxyl ester. In some embodiments, R ¹⁵ is acyl or acyloxy. In some embodiments, R ¹⁵ is acylamino or aminoacyl. In some embodiments,
R ¹⁵ is alkylamide or substituted alkylamide. In some embodiments, R
¹⁵ is sulfonyl. In some embodiments, R ¹⁵ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ¹⁵ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. be. In certain embodiments, R ¹⁵ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅
substituted heteroaryl, or _C6 heteroaryl or _C6 substituted heteroaryl. In certain embodiments, R ¹⁵ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, or C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C
_3-5 substituted cycloalkyl. In some embodiments, R ¹⁵ is heterocyclyl or substituted heterocyclyl, such as C _3-8 heterocyclyl or C _3-8 substituted heterocyclyl, C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5
heterocyclyl or C _3-5 substituted heterocyclyl.

In some embodiments, each R ¹⁵ is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl , cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. In these embodiments, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxylester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,
Substituted cycloalkyl, heterocyclyl and substituted heterocyclyl substituents are defined above at R ¹⁵
as described for

In some embodiments, the tether group includes an acetal group, disulfide, hydrazine or ester. In some embodiments,
The linking chain group includes an acetal group. In some embodiments, the linking chain group includes a disulfide. In some embodiments, the linking chain group includes hydrazine. In some embodiments, the linking chain group includes an ester.

As noted above, in some embodiments, L is -(T ¹ -V ¹ ) _a -(T ² -V
² ) a linker comprising _b- (T ³ -V ³ ) _c -(T ⁴ -V ⁴ ) _d -, where a, b
, c and d are each independently 0 or 1, and the sum of a, b, c and d is 1
~4.

In some embodiments, in the linker
T ¹ is selected from (C ₁ -C ₁₂ )alkyl and substituted (C ₁ -C ₁₂ )alkyl;
T ² , T ³ and T ⁴ are each independently (C ₁ -C ₁₂ )alkyl, substituted (C
₁ -C ₁₂ )alkyl, (EDA) _w , (PEG) _n , (AA) _p , —(CR ¹³ OH)
_h -, 4-amino-piperidine (4AP), acetal groups, hydrazines and esters; and V ¹ , V ² , V ³ and V ⁴ are each independently a covalent bond, -CO-, - NR ^1
5 -, -NR ¹⁵ (CH ₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -N
R ¹⁵ CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —S
selected from the group consisting of O ₂ —, —SO ₂ NR ¹⁵ —, —NR ¹⁵ SO ₂ — and —P(O)OH—, wherein q is an integer from 1 to 6;
here,
(PEG) _n is

where n is an integer from 1 to 30;
EDA has the following structure:

wherein y is an integer from 1 to 6 and r is 0 or 1;
4-amino-piperidine (4AP) is

is;
AA is an amino acid residue, where p is an integer from 1-20;
each R ¹⁵ and R ¹² is independently selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl, wherein any two adjacent R ¹² groups are cyclically linked to form a piperazinyl ring and R ¹³ is selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl.

In some embodiments, T ¹ , T ² , T ³ and T ⁴ and V ¹ , V ² , V ³ and V ⁴ are selected from the table below, eg, one row of the table below.

In some embodiments, L is -(L ¹ ) _a -(L ² ) _b -(L ³ ) _c -(L ⁴ ) _d
-, wherein -(L ¹ ) _a - is -(T ¹ -V ¹ ) _a - and -(
L ² ) _b - is -(T ² -V ² ) _b -, -(L ³ ) _c - is -(T ³ -V ³ ) _c -, -(L ⁴ ) _d - is -( T ⁴ -V ⁴ ) _d -.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (AA) _p , V ² is -NR ¹⁵ -, T ³ is (PEG) _n , V ³ is -CO-, T ⁴ is absent and V ⁴ is absent.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (EDA) _w , V ² is -CO-, T ³ is (CR ¹³ OH) _h , V ³ is —CONR ¹⁵ —, T ⁴ is (C ₁ -C ₁₂ )alkyl and V ⁴ is —CO—.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (AA) _p , V ² is -NR ¹⁵ -, T ³ is (C ₁ -C ₁₂ )alkyl, V ³ is —CO—, T ⁴ is absent and V ⁴ is absent.

In some embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl and V ¹ is -CONR
¹⁵ -, T ² is (PEG) _n , V ² is -CO-, T ³ is absent, V
³ is not present, T ⁴ is not present, V ⁴ is not present.

In some embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is —CO—, T ² is (AA) _p , V ² is absent, T ³ is present does not exist ^, V3 does not exist, and T
⁴ does not exist and ^V4 does not exist.

In some embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl and V ¹ is -CONR
¹⁵ -, T ² is (PEG) _n , V ² is -NR ¹⁵ -, T ³ is absent, V ³ is absent, T ⁴ is absent, V ⁴ is present do not do.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (AA) _p , V ² is -NR ¹⁵ -, T ³ is (PEG) _n , V ³ is -NR ¹⁵ -, T ⁴ is absent and V ⁴ is absent.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (EDA) _w , V ² is -CO-, T ³ is not present, V ³ is not present, T ⁴ is not present, V ⁴ is not present.

In some embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl and V ¹ is -CONR
¹⁵ —, T ² is (C ₁ -C ₁₂ )alkyl, V ² is —NR ¹⁵ —, T
³ is not present, V ³ is not present, T ⁴ is not present, V ⁴ is not present.

In some embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl and V ¹ is -CONR
¹⁵ −, T ² is (PEG) _n , V ² is —CO—, T ³ is (EDA) _w
, V3 does not exist ^{, T4} does not exist, and ^V4 does not exist.

In some embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is —CO—, T ² is (EDA) _w , V ² is absent and T ³ is present. does not exist ^, V3 does not exist,
^T4 is absent and ^V4 is absent.

In some embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl and V ¹ is -CONR
¹⁵ -, T ² is (PEG) _n , V ² is -CO-, T ³ is (AA) _p , V ³ is absent, T ⁴ is absent, V ⁴ does not exist.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (EDA) _w , V ² is -CO-, T ³ is (CR ¹³ OH) _h , V ³ is -CO-, T ⁴ is (AA) _p and V ⁴ is absent.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (AA) _p , V ² is -NR ¹⁵ -, T ³ is (C ₁ -C ₁₂ )alkyl, V ³ is —CO—, T ⁴ is (AA) _p and V ⁴ is absent.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (AA) _p , V ² is -NR ¹⁵ -, T ³ is (PEG) _n , V ³ is -CO-, T ⁴ is (AA) _p and V ⁴ is absent.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (AA) _p , V ² is -NR ¹¹ -, T ³ is (PEG) _n , V ³ is -SO ₂ -, T ⁴ is (AA) _p and V ⁴ is absent.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (EDA) _w , V ² is -CO-, T ³ is (CR ¹³ OH) _h , V ³ is -CONR ¹⁵ -, T ⁴ is (PEG) _n and V ⁴ is -CO-.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (CR ¹³ OH) _h , V ² is -CO-, T ³ is absent and V ³
is absent, T ⁴ is absent, and V ⁴ is absent.

In some embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl and V ¹ is -CONR
¹⁵ —, T ² is substituted (C ₁ -C ₁₂ )alkyl, V ² is —NR ¹⁵ —, T ³ is (PEG) _n , V ³ is —CO—, T ⁴ does not exist and ^V4 does not exist.

In some embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl and V ¹ is -SO ₂ -
, T ² is (C ₁ -C ₁₂ )alkyl, V ² is —CO—, T ³ is absent, V ³ is absent, T ⁴ is absent, V ⁴ is not exist.

In some embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl and V ¹ is -CONR
¹⁵ —, T ² is (C ₁ -C ₁₂ )alkyl, V ² is absent, T ³ is (CR
¹³ OH) _h , V ³ is -CONR ¹⁵ -, T ⁴ is absent and V ⁴ is absent.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (AA) _p , V ² is -NR ¹⁵ -, T ³ is (PEG) _n , V ³ is -CO-, T ⁴ is (AA) _p and V ⁴ is -NR ¹⁵ -.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (AA) _p , V ² is -NR ¹⁵ -, T ³ is (PEG) _n , V ³ is -P(O)OH-, T ⁴ is (AA) _p and V ⁴ is absent.

In some embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is —CO—, T ² is (EDA) _w , V ² is absent, and T ³ is ( ^AA ) _p , V3 absent, ^T4 absent, ^V4 absent.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (EDA) _w , V ² is -CO-, T ³ is (CR ¹³ OH) _h , V ³ is —CONR ¹⁵ —, T ⁴ is (C ₁ -C ₁₂ )alkyl and V ⁴ is —CO(AA) 2 _p .

In some embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl and V ¹ is -CONR
¹⁵ —, T ² is (C ₁ -C ₁₂ )alkyl, V ² is —NR ¹⁵ —, T
³ is absent, V ³ is -CO-, T ⁴ is absent, V ⁴ is absent.

In some embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl and V ¹ is -CONR
¹⁵ —, T ² is (C ₁ -C ₁₂ )alkyl, V ² is —NR ¹⁵ —, T
³ is absent, V ³ is -CO-, T ⁴ is (C ₁ -C ₁₂ )alkyl and V ⁴ is -NR ¹⁵ -.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is (EDA) _w , V ² is -CO-, T ³ is (CR ¹³ OH) _h , V ³ is —CONR ¹⁵ —, T ⁴ is (PEG) _n , V ⁴ is —CO(AA
) is _p .

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is 4AP, V ² is -CO-, T ³ is (C ₁ -C ₁₂ )alkyl, V ³ is —CO—, T ⁴ is (AA) _p and V ⁴ is absent.

In certain embodiments, T ¹ is (C ₁ -C ₁₂ )alkyl, V ¹ is -CO-, T ² is 4AP, V ² is -CO-, T ³ is (C ₁ -C ₁₂ )alkyl, V ³ is —CO—, T ⁴ is absent and V ⁴ is absent.

In some embodiments, the linker is represented by one of the structures below.

In some embodiments of the above linker structures, each f is independently 0 or 1-12
each y is independently an integer from 0 or 1 to 20; each n is independently
is 0 or an integer from 1 to 30; each p is independently 0 or an integer from 1 to 20; each h is independently 0 or an integer from 1 to 12; independently hydrogen, alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide,
sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl; and each R' is independently H, side of the amino acid chain group,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
Alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acylamino, aminoacyl, alkylamido, substituted alkylamido, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl , cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. In some embodiments of the above linker structures, each f is independently 0, 1, 2, 3, 4, 5 or 6; each y is independently 0
, 1, 2, 3, 4, 5 or 6; each n is independently 0, 1, 2, 3, 4, 5 or 6; each p is independently 0, 1 , 2, 3, 4, 5 or 6; and each h is independently 0, 1, 2, 3, 4, 5 or 6. In certain embodiments of the above linker structures, each R is independently H, methyl or -( _CH2 ) _m -OH, where m is 1, 2, 3 or 4 (e.g., 2).

In some embodiments of linker L, T ¹ is (C ₁ -C ₁₂ )alkyl and V ¹
is -CO-, T ² is 4AP, V ² is -CO-, T ³ is (C ₁ -C ₁₂
) alkyl, V ³ is —CO—, T ⁴ is absent and V ⁴ is absent. In some embodiments, T ¹ is ethylene, V ¹ is -CO-, T ² is 4AP, V ² is -CO-, T ³ is ethylene, V ³ is -CO −, T ⁴ is absent and V ⁴ is absent. In some embodiments, T ¹ is ethylene and V ¹ is -CO
-, T ² is 4AP, V ² is -CO-, T ³ is ethylene, V ³ is -CO-, T ⁴ is absent, V ⁴ is absent, wherein T ² (eg, 4AP) has the following structure:

where R ¹² is a polyethylene glycol moiety (eg, polyethylene glycol or modified polyethylene glycol).

In some embodiments, the linker L has the structure:

(wherein each f is independently an integer from 1 to 12 and n is an integer from 1 to 30).

In some embodiments, f is 1. In some embodiments, f is two. In some embodiments, one of f is two and one of f is one.

In some embodiments, n is 1.

In certain embodiments, the left side of said linker structure is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety and the right side of said linker structure is attached to maytansine.

Any of the chemicals, linkers and coupling moieties shown in the structures above can be adapted for use in the present compounds and conjugates.

Further disclosure regarding methods of making hydrazinyl-indolyl and hydrazinyl-pyrrolo-pyridinyl compounds and conjugates can be found in US Patent Application Publication Nos. 2014/0141025 filed March 11, 2013 and November 26, 2014. US Patent Application Publication No. 2015/0157736, the disclosures of each of which are incorporated herein by reference.

Anti-CD22 Antibodies As noted above, the conjugate can include an anti-CD22 antibody as substituent W2, wherein the anti-CD22 antibody is modified to include a ² -formylglycine (FGly) residue. ing. Amino acids used herein may be referred to by their standard names, their standard three-letter abbreviations and/or their standard one-letter abbreviations, such as: Alanine or Ala or A cysteine or Cys or C; aspartic acid or Asp or D; glutamic acid or Glu or E; phenylalanine or Phe or F; glycine or Gly or G; Leucine or Leu or L; Methionine or Met or M; Asparagine or Asn or N
glutamine or Gln or Q; arginine or Arg or R; serine or Ser or S; threonine or Thr or T; valine or Val or V;

In some cases, a suitable anti-CD22 antibody specifically binds to a CD22 polypeptide, wherein the epitope is an amino acid residue within the CD22 antigen (eg, the CD22 amino acid sequences shown in FIGS. 8A-8C). Within amino acids 1-847 (1-847) of, amino acids 1-
759, within amino acids 1-751, or within amino acids 1-670).

The CD22 epitope is at least about 75%, at least about 80%, at least about 85% over a continuous stretch of about 500 amino acids to about 670 amino acids of the amino acid sequence of human CD22 isoform 4 shown in FIGS. , at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity. The CD22 epitope is at least about 75%, at least about 80%, at least about 8%, 80%, 80%, 80%, 80%, 80%, 80%, 80%, 80%, 80%, or 80%, continuous stretches of about 500 amino acids to about 751 amino acids of the amino acid sequence of human CD22 isoform 3 shown in Figures 8A-8C.
5%, at least about 90%, at least about 95%, at least about 98%, at least about 9
Polypeptides having 9% or 100% amino acid sequence identity can be formed. C.
The D22 epitope is at least about 75 to a continuous stretch of about 500 amino acids to about 759 amino acids of the amino acid sequence of human CD22 isoform 2 shown in Figures 8A-8C.
%, at least about 80%, at least about 85%, at least about 90%, at least about 95
%, at least about 98%, at least about 99% or 100% amino acid sequence identity. The CD22 epitope is at least about 75%, at least about 80%, at least about 85% over a continuous stretch of about 500 amino acids to about 847 amino acids of the human CD22 isoform 1 amino acid sequence shown in FIGS.
, at least about 90%, at least about 95%, at least about 98%, at least about 99%
or formed by polypeptides with 100% amino acid sequence identity.

A "CD22 antigen" or "CD22 polypeptide" refers to about 500 amino acids (aa
) to about 847 aa (isoform 1), to about 759 aa (isoform 2), to about 7
at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99 for a continuous stretch of 51 aa (isoform 3) or to about 670 aa (isoform 4) Amino acid sequences having % or 100% amino acid sequence identity can be included.

In some cases, suitable anti-CD22 antibodies exhibit high affinity binding to CD22. For example, in some cases, suitable anti-CD22 antibodies are at least about 10
⁻⁷ M, at least about 10 ⁻⁸ M, at least about 10 ⁻⁹ M, at least about 10 ⁻¹⁰ M
, binds CD22 with an affinity of at least about 10 ⁻¹¹ M or at least about 10 ⁻¹² M or greater than about 10 ⁻¹² M. In some cases, a suitable anti-CD22 antibody is about 10 ⁻⁷ M to about 10 ⁻⁸ M, about 10 ⁻⁸ M to about 10 ⁻⁹ M, about 10 ⁻⁹ M to about 10 ⁻
Binds an epitope present on CD22 with an affinity of ¹⁰ M, from about 10 ⁻¹⁰ M to about 10 ⁻¹¹ M, or from about 10 ⁻¹¹ M to about 10 ⁻¹² M, or greater than 10 ⁻¹² M.

In some cases, the appropriate anti-CD22 antibody competes with a second anti-CD22 antibody for binding to an epitope within CD22 and/or the same CD2 antibody as the second anti-CD22 antibody.
Binds epitopes within 2. In some cases, an anti-CD22 antibody that competes with a second anti-CD22 antibody for binding to an epitope within CD22 also binds to the same epitope as the second anti-CD22 antibody. In some cases, an anti-CD22 antibody that competes with a second anti-CD22 antibody for binding to an epitope within CD22 binds to an epitope that overlaps with the epitope bound by the second anti-CD22 antibody. In some cases, anti-CD
22 antibodies have been humanized.

In some cases, suitable anti-CD22 antibodies can induce apoptosis in cells that express CD22 on the cell surface.

In some cases, an anti-CD22 antibody suitable for use in the conjugate is
inhibiting the proliferation of human tumor cells that overexpress CD22, wherein the inhibition is in vitro;
Occurs in vivo or both in vitro and in vivo. For example, in some cases, anti-CD22 antibodies suitable for use in the present conjugates reduce proliferation of human tumor cells that overexpress CD22 by at least about 15%, at least about 20%, at least about 25%
, at least about 30%, at least about 40%, at least about 50%, at least about 60%
, at least about 70%, at least about 80% or more than 80%, such as at least about 8
5%, at least about 90%, at least about 95%, at least about 98%, at least about 9
9% or 100% suppression.

In some cases, a suitable anti-CD22 antibody has a CD22 epitope [e.g., C
within the D22 antigen (eg, amino acid 1 of the CD22 amino acid sequence shown in FIGS. 8A-8C)
-847, within amino acids 1-759, within amino acids 1-751 or within amino acids 1-670)], for
SEQ ID NO//), YISSGGGTTYYPDTVKG (VH CDR2; SEQ ID NO//
) and HSGYGSSYGVLFAY (VH CDR3; SEQ ID NO://). In some cases, the anti-CD22 antibody is humanized. In some cases, a suitable anti-CD22 antibody is
CD22 epitopes [e.g., within the CD22 antigen (e.g., C shown in Figures 8A-8C
Within amino acids 1-847, within amino acids 1-759, amino acids 1-75 of the D22 amino acid sequence
RASQDISNYLN (VL CDR1; SEQ ID NO://), YTSILHS (VL
CDR2; SEQ ID NO//) and QQGNTLPWT (VL CDR3; SEQ ID NO//)
compete with antibodies containing light chain CDRs selected from In some cases, the anti-CD2
2 antibodies have been humanized.

In some cases, a suitable anti-CD22 antibody has a CD22 epitope [e.g., C
within the D22 antigen (eg, amino acid 1 of the CD22 amino acid sequence shown in FIGS. 8A-8C)
-847, within amino acids 1-759, within amino acids 1-751 or within amino acids 1-670)], for
H CDR1; SEQ ID NO //), YISSGGGTTYYPDTVKG (VH CDR2
; SEQ ID NO//) and HSGYGSSYGVLFAY (VH CDR3; SEQ ID NO//
) to compete with antibodies containing In some cases, the anti-CD22 antibody is humanized. In some cases, a suitable anti-CD22 antibody has a CD22 epitope [e.g.
within the CD22 antigen (eg, within amino acids 1-847, within amino acids 1-759, within amino acids 1-751 or within amino acids 1-670 of the CD22 amino acid sequences shown in FIGS. 8A-8C)
VL CDR RASQDI for binding to an epitope comprising amino acid residues
Compete with antibodies including SNYLN (VL CDR1; SEQ ID NO//), YTSILHS (VL CDR2; SEQ ID NO//) and QQGNTLPWT (VL CDR3; SEQ ID NO//). In some cases, the anti-CD22 antibody is humanized. In some cases, a suitable anti-CD22 antibody is a CD22 epitope [e.g., within the CD22 antigen (
For example, binding to an epitope comprising amino acid residues within amino acids 1-847, within amino acids 1-759, within amino acids 1-751, or within amino acids 1-670 of the CD22 amino acid sequence shown in FIGS. Regarding VH CDR IYDMS (VH CDR1;
SEQ ID NO//), YISSGGGTTYYPDTVKG (VH CDR2; SEQ ID NO//
) and HSGYGSSYGVLFAY (VH CDR3; SEQ ID NO://) and VL
CDR RASQDISNYLN (VL CDR1; SEQ ID NO://), YTSILHS
(VL CDR2; SEQ ID NO//) and QQGNTLPWT (VL CDR3; SEQ ID NO//). In some cases, the anti-CD22 antibody is humanized.

In some cases, a suitable anti-CD22 antibody is VH CDR IYDMS (VH
CDR1; SEQ ID NO//), YISSGGGTTYYPDTVKG (VH CDR2; SEQ ID NO//) and HSGYGSSYGVLFAY (VH CDR3; SEQ ID NO//). In some cases, the anti-CD22 antibody is humanized. Appropriate anti-CD2
2 antibodies are VL CDR RASQDISNYLN (VL CDR1; SEQ ID NO://), Y
TSILHS (VL CDR2; SEQ ID NO://) and QQGNTLPWT (VL CD
R3; SEQ ID NO//). In some cases, the anti-CD22 antibody is humanized. In some cases, a suitable anti-CD22 antibody is the VH CDR IYDMS (
VH CDR1; SEQ ID NO //), YISSGGGTTYYPDTVKG (VH CDR
2; SEQ ID NO//) and HSGYGSSYGVLFAY (VH CDR3; SEQ ID NO/
/) and VL CDR RASQDISNYLN (VL CDR1; SEQ ID NO://)
, YTSILHS (VL CDR2; SEQ ID NO: //) and QQGNTLPWT (VL
CDR3; SEQ ID NO//). In some cases, the anti-CD22 antibody is humanized.

In some cases, a suitable anti-CD22 antibody has the following amino acid sequence: EVQLV
ESGGGLVKPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGL
EWVAYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMSS
Contains VH CDRs present within the anti-CD22 VH region including LRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS (SEQ ID NO://). In some cases, the anti-CD22 antibody is humanized.

In some cases, a suitable anti-CD22 antibody has the following amino acid sequence: DIQMT
QSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVK
LLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQQEDFAT
Anti-CD22 containing YFCQQGNTLPWTFGGGTKVEIKR (SEQ ID NO://)
Contains the VL CDRs that reside within the VL region. In some cases, the anti-CD22 antibody is humanized.

In some cases, a suitable anti-CD22 antibody is EVQLVESGGGLVKPG
GSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGG
GTTYYPDTVKGRFTISRDNAKNSLYLQMSSLRAEDTAMYY
VH CDRs present in CARHSGYGSSYGVLFAYWGQGTLVTVSS (SEQ ID NO://) and DIQMTQSPSSLSASVGDRVTITCRASQDIS
NYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTD
Contains the VL CDRs present in YTLTISSLQQEDFATYFCQQGNTLPWTFGGGTKVEIKR (SEQ ID NO://). In some cases, the anti-CD22 antibody is humanized.

In some cases, a suitable anti-CD22 antibody has a) the amino acid sequence EVQLVES
GGGLVKPGGSLX ¹ LSCAASGFAFSIYDMSWVRQAPGKGLE
^{WVAYISSGGGTTYYPDTVKGRFTISRDNAKNX2LYLQMX3 _}
SLRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS(
SEQ ID NO: 1) [where X ¹ is K (Lys) or R (Arg); X ² is S (Se
r) or T (Thr); and X ³ is N (Asn) or S (Ser)]
and b) an immunoglobulin light chain.

The light chain can have any suitable _VL amino acid sequence as long as the resulting antibody specifically binds to CD22.

A typical _VL amino acid sequence is
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQ
KPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSL
QQEDFATYFCQQGNTLPWTFGGGTKVEIKR (SEQ ID NO: 7; VK1
);
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQ
KPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSL
QPEDFATYFCQQGNTLPWTFGGGTKVEIKR (SEQ ID NO: 8; VK2
); and DIQMTQSPSSVSASVGDRVTITCRASQDISNYLNWYQQ
KPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSL
QPEDFATYFCQQGNTLPWTFGGGTKVEIKR (SEQ ID NO: 9; VK4
)
including.

Thus, for example, a suitable anti-CD22 antibody can comprise a) a heavy chain comprising a VH region having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain comprising a VL region of VK1. In other cases, a suitable anti-CD22 antibody may comprise a) a heavy chain comprising a VH region having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain comprising a VL region of VK2. In still other cases, the anti-CD22 antibody can comprise a) a heavy chain comprising a VH region having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain comprising a VL region of VK4.

In some cases, a suitable anti-CD22 antibody has a) amino acid sequence: DIQMTQ
SPSSX ¹ SASVGDRVTITCRASQDISNYLNWYQQKPGKAX ^{2
KLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQX3EDF}
ATYFCQQGNTLPWTFGGGTKVEIK (SEQ ID NO: 2) [where X ¹ is L
(Leu) or V(Val); X2 is V ⁽ Val) or P(Pro);
and X ³ is Q (Gln) or P (Pro)]; and b
) immunoglobulin heavy chains. The heavy chain is
EVQLVESGGGGLVKPGGSLKLSCAASGFAFSIYDMSWVR
QAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNT
LYLQMSSLRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTL
VTVSS (SEQ ID NO: 3; VH3);
EVQLVESGGGGLVKPGGSLRLSCAASGFAFSIYDMSWVR
QAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNS
LYLQMSSLRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTL
VTVSS (SEQ ID NO: 4; VH4);
EVQLVESGGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQ
APGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNSL
YLQMNSLRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLV
TVSS (SEQ ID NO: 5; VH5); and EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQ
APGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNSL
YLQMSSLRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLV
TVSS (SEQ ID NO: 6; VH6)
may comprise an amino acid sequence selected from

In some cases, a suitable anti-CD22 antibody has the following amino acid sequence: EVQLV
ESGGGLVKPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGL
EWVAYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMSS
LRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS (SEQ ID NO://).

In some cases, a suitable anti-CD22 antibody has the following amino acid sequence: EVQLV
ESGGGLVKPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGL
EWVAYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMSS
VH region comprising LRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS (SEQ ID NO: //) and the following amino acid sequence: DIQMTQSPSSLSASV
GDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSILH
SGVPSRFSGSGSGTDYTLTISSLQQEDFATYFCQQGNTLP
and a VL region containing WTFGGGTKVEIKR (SEQ ID NO://).

Modified Constant Region Sequences As noted above, the amino acid sequence of an anti-CD22 antibody may be modified in vivo (e.g., in cells by a
during translation of ld-tagged proteins) or in vitro (e.g., in cell-free systems)
Formylglycine synthase (by contacting the d-tag containing protein with FGE)
It is modified to contain a sulfatase motif containing a serine or cysteine residue that can be converted (oxidized) to a 2-formylglycine (FGly) residue by the action of FGE).
Such sulfatase motifs may also be referred to herein as FGE modification sites.

Sulfatase Motifs The minimal sulfatase motif of the aldehyde tag is usually 5 or 6 amino acid residues long, usually 6 amino acid residues or less. The sulfatase motif provided in the Ig polypeptide is at least 5 or 6 amino acid residues, 16, 15, 14, 1
To define a sulfatase motif of less than 3, 12, 11, 10, 9, 8 or 7 amino acid residues in length, for example 5-16, 6-16, 5-15, 6-15, 5-14 , 6
~14, 5~13, 6~13, 5~12, 6~12, 5~11, 6~11, 5~10, 6
It can be ~10, 5-9, 6-9, 5-8 or 6-8 amino acid residues long.

In certain embodiments, the polypeptide of interest includes one or more amino acid residues relative to the native amino acid sequence, such as 2 or more, or 3 or more, or 4 or more, to obtain a sequence of sulfatase motifs in the polypeptide. , or 5 or more, or 6 or more, or 7
or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19 or more, Or those in which 20 or more amino acid residues have been inserted, deleted, or substituted. In certain embodiments, the polypeptide is
20, 19, 18, 17 of the amino acid sequence relative to the native amino acid sequence of the polypeptide;
modification (insertion, addition, deletion and/or substitution) of no more than 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues include. If the amino acid sequence unique to the polypeptide (eg, an anti-CD22 antibody) contains one or more residues of the desired sulfatase motif, the total number of residue modifications is, for example, site-specific modification of amino acid residues adjacent to the natural amino acid residue (
insertions, additions, deletions, substitutions). In certain embodiments, targeted anti-CD2
2 The degree of modification of the native amino acid sequence of a polypeptide is minimized to minimize the number of amino acid residues inserted, deleted, substituted or added (eg, to the N- or C-termini). Minimizing the degree of amino acid sequence modification of the target anti-CD22 polypeptide can minimize the effects that such modifications can have on anti-CD22 function and/or structure.

Aldehyde tags of particular interest are those that include at least a minimal sulfatase motif (also referred to as a "consensus sulfatase motif"), although longer aldehyde tags are also envisioned and encompassed by the present disclosure and are useful in the compositions and methods of the present disclosure. It should be noted that it is readily understood that it can be Thus, the aldehyde tag can contain a minimal sulfatase motif of 5 or 6 residues, or it can be longer, with additional amino acid residues flanking the N-terminus and/or C-terminus of the motif. may contain a minimal sulfatase motif capable of being For example, an aldehyde tag of 5 or 6 amino acid residues and 5, 6, 7, 8, 9, 10, 11, 12, 13,
Aldehyde tags of longer amino acid sequences of 14, 15, 16, 17, 18, 19, 20 or more amino acid residues are also envisioned.

The aldehyde tag can be at or near the C-terminus of the Ig heavy chain. For example, the aldehyde tag can be within the C-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of a native wild-type Ig heavy chain. An aldehyde tag can be present within the CH1 domain of an Ig heavy chain. An aldehyde tag can be present within the CH2 domain of an Ig heavy chain. An aldehyde tag can be present within the CH3 domain of the Ig heavy chain. Aldehyde tags may be present in Ig light chain constant regions, such as the kappa light chain constant region or the lambda light chain constant region.

In certain embodiments, the sulfatase motif used has the formula:
X ¹ Z ¹⁰ X ² Z ²⁰ X ³ Z ³⁰ (I′)
can be denoted by, where
Z ¹⁰ is cysteine or serine (which can also be represented by (C/S));
Z ²⁰ is either a proline or alanine residue (which can also be represented by (P/A));
Z ³⁰ can be a basic amino acid (e.g. arginine (R), and lysine (K) or histidine (H), e.g. lysine) or an aliphatic amino acid (alanine (A), glycine (G), leucine (L) , valine (V), isoleucine (I) or proline (P
), such as A, G, L, V or I);
X ¹ may or may not be present, and if present, any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid), such as can be L, M, V, S or T, such as L, M, S or V, but X ¹ is present if the sulfatase motif is present at the N-terminus of the target polypeptide;
X ² and X ³ can independently be any amino acid, but usually aliphatic, polar uncharged or sulfur-containing amino acids (i.e. other than aromatic or charged amino acids) such as S, T, A, V, G or C, for example S, T, A, V or G.

The amino acid sequence of the anti-CD22 heavy and/or light chain has the formula: X ¹ Z ¹⁰ X ² Z ²⁰ X ³
can be modified to give a sequence of at least 5 amino acids of Z ³⁰ , wherein
Z ¹⁰ is cysteine or serine;
^Z20 is a proline or alanine residue;
Z ³⁰ is an aliphatic or basic amino acid;
X ¹ may or may not be present and, if present, any amino acid, except that X ¹ is present if a heterologous sulfatase motif is present at the N-terminus of said polypeptide;
X ² and X ³ are each independently any amino acid;
The sequence is within or adjacent to the solvent accessible loop region of the Ig constant region and the sequence is not at the C-terminus of the Ig heavy chain.

The sulfatase motif is generally the FGE of choice (e.g., the FGE present in the host cell in which the aldehyde-tagged polypeptide is expressed, or the FGE that will be contacted with the aldehyde-tagged polypeptide in a cell-free in vitro method). ) so that it can be transformed by

For example, if the FGE is a eukaryotic FGE (eg mammalian FGE, eg human FGE), the sulfatase motif has the formula:
^{X1CX2PX3Z30 ( I '} ')
, where
X ¹ may or may not be present, and if present, any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid), such as can be L, M, S or V, but X ¹ is present if a sulfatase motif is present at the N-terminus of the target polypeptide;
X ² and X ³ are independently any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid)
, such as S, T, A, V, G or C, such as S, T, A, V or G;
Z ³⁰ can be a basic amino acid (e.g. arginine (R), and lysine (K) or histidine (H), e.g. lysine) or an aliphatic amino acid (alanine (A), glycine (G), leucine (L) , valine (V), isoleucine (I) or proline (P
), such as A, G, L, V or I).

Specific examples of sulfatases include LCTPSR (SEQ ID NO: //), MCTPSR (SEQ ID NO: //), VCTPSR (SEQ ID NO: //), LCSPSR (SEQ ID NO: //), LCAPS
R (SEQ ID NO//), LCVPSR (SEQ ID NO//), LCGPSR (SEQ ID NO//),
ICTPAR (SEQ ID NO: //), LCTPSK (SEQ ID NO: //), MCTPSK (SEQ ID NO: //), VCTPSK (SEQ ID NO: //), LCSPSK (SEQ ID NO: //), LCAPS
K (SEQ ID NO//), LCVPSK (SEQ ID NO//), LCGPSSK (SEQ ID NO//),
LCTPSA (SEQ ID NO: //), ICTPAA (SEQ ID NO: //), MCTPSA (SEQ ID NO: //), VCTPSA (SEQ ID NO: //), LCSPSA (SEQ ID NO: //), LCAPS
A (SEQ ID NO//), LCVPSA (SEQ ID NO//) and LCGPSA (SEQ ID NO//
) is included.

Upon action of FGE on FGly-containing sequence -modified anti-CD22 heavy and/or light chains, a serine or cysteine within the sulfatase motif is converted to FGly. Therefore, FGly
The containing sulfatase motif has the formula:
X ¹ (FGly) X ² Z ²⁰ X ³ Z ³⁰ (I''')
, where
FGly is a formylglycine residue;
Z ²⁰ is either a proline or alanine residue (which can also be represented by (P/A));
Z ³⁰ can be a basic amino acid (e.g. arginine (R), and lysine (K) or histidine (H), usually lysine) or an aliphatic amino acid (alanine (A), glycine (G), leucine (L ), valine (V), isoleucine (I) or proline (P
), such as A, G, L, V or I);
X ¹ may or may not be present, and if present, any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid), such as can be L, M, V, S or T, such as L, M, S or V, but X ¹ is present if the sulfatase motif is present at the N-terminus of the target polypeptide;
X ² and X ³ are independently any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid)
, such as S, T, A, V, G or C, such as S, T, A, V or G.

As described above, a modified polypeptide containing an FGly residue can be prepared by reacting FGly with a drug (e.g., a drug containing a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety described above) (e.g., maytansinoids) to give a FGly'-containing sulfatase motif. As used herein, the term FGly' refers to a modified amino acid residue (eg, modified amino acid residue of formula (I)) of a sulfatase motif that is coupled to a drug such as maytansinoid. Thus, the FGly'-containing sulfatase motif has the formula:
X ¹ (FGly') X ² Z ²⁰ X ³ Z ³⁰ (II)
, where
FGly' is a modified amino acid residue of formula (I);
Z ²⁰ is either a proline or alanine residue (which can also be represented by (P/A));
Z ³⁰ can be a basic amino acid (e.g. arginine (R), and lysine (K) or histidine (H), usually lysine) or an aliphatic amino acid (alanine (A), glycine (G), leucine (L ), valine (V), isoleucine (I) or proline (P
), such as A, G, L, V or I);
X ¹ may or may not be present, and if present, any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid), such as can be L, M, V, S or T, such as L, M or V, but if the sulfatase motif is at the N-terminus of the target polypeptide,
X ¹ is present;
X ² and X ³ are independently any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid)
, such as S, T, A, V, G or C, such as S, T, A, V or G.

In certain embodiments, the modified amino acid residue of formula (I) is located at the C-terminus of the heavy chain constant region of the anti-CD22 antibody. In some cases, the heavy chain constant region has the formula:
X ¹ (FGly') X ² Z ²⁰ X ³ Z ³⁰ (II)
contains an array of , where
FGly' is a modified amino acid residue of formula (I);
Z ²⁰ is either a proline or alanine residue (which can also be represented by (P/A));
Z ³⁰ can be a basic amino acid (e.g. arginine (R), and lysine (K) or histidine (H), usually lysine) or an aliphatic amino acid (alanine (A), glycine (G), leucine (L ), valine (V), isoleucine (I) or proline (P
), such as A, G, L, V or I);
X ¹ may or may not be present, and if present, any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid), such as can be L, M, V, S or T, such as L, M or V, but if the sulfatase motif is at the N-terminus of the target polypeptide,
X ¹ is present;
X ² and X ³ are independently any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid)
, such as S, T, A, V, G or C, such as S, T, A, V or G;
The sequence is C-terminal to the amino acid sequence QKSLSLSPGK, and the sequence may comprise 1, 2, 3, 4, 5, or 5-10 amino acids not present in the native wild-type Ig heavy chain constant region. .

In certain embodiments, the heavy chain constant region has the sequence SLSLSPGSL (FGly
') contains TPSRGS.

In certain embodiments, the modified amino acid residue of formula (I) is located in the light chain constant region of the anti-CD22 antibody. In some embodiments, the light chain constant region has the formula:
X ¹ (FGly') X ² Z ²⁰ X ³ Z ³⁰ (II)
contains an array of , where
FGly' is a modified amino acid residue of formula (I);
Z ²⁰ is either a proline or alanine residue (which can also be represented by (P/A));
Z ³⁰ can be a basic amino acid (e.g. arginine (R), and lysine (K) or histidine (H), usually lysine) or an aliphatic amino acid (alanine (A), glycine (G), leucine (L ), valine (V), isoleucine (I) or proline (P
), such as A, G, L, V or I);
X ¹ may or may not be present, and if present, any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid), such as can be L, M, V, S or T, such as L, M or V, but if the sulfatase motif is at the N-terminus of the target polypeptide,
X ¹ is present;
X ² and X ³ are independently any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid)
, such as S, T, A, V, G or C, such as S, T, A, V or G;
The sequence is present on the C-terminal side of the amino acid sequence KVDNAL (SEQ ID NO://), and/
or on the N-terminal side of the amino acid sequence QSGNSQ (SEQ ID NO://).

In some embodiments, the light chain constant region has the sequence KVDNAL(FGly')TPSR
Contains QSGNSQ (SEQ ID NO://).

In certain embodiments, the modified amino acid residue of formula (I) is the heavy chain CH1 of the anti-CD22 antibody.
located in the area. In certain embodiments, the heavy chain CH1 region has the formula:
X ¹ (FGly') X ² Z ²⁰ X ³ Z ³⁰ (II)
contains an array of , where
FGly' is a modified amino acid residue of formula (I);
Z ²⁰ is either a proline or alanine residue (which can also be represented by (P/A));
Z ³⁰ can be a basic amino acid (e.g. arginine (R), and lysine (K) or histidine (H), usually lysine) or an aliphatic amino acid (alanine (A), glycine (G), leucine (L ), valine (V), isoleucine (I) or proline (P
), such as A, G, L, V or I);
X ¹ may or may not be present, and if present, any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid), such as can be L, M, V, S or T, such as L, M or V, but if the sulfatase motif is at the N-terminus of the target polypeptide,
X ¹ is present;
X ² and X ³ are independently any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid or a polar uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid)
, such as S, T, A, V, G or C, such as S, T, A, V or G;
The sequence is present on the C-terminal side of the amino acid sequence SWNSGA (SEQ ID NO://), and/
Alternatively, it exists on the N-terminal side of the amino acid sequence GVHTFP (SEQ ID NO://).

In some embodiments, the heavy chain CH1 region has the sequence SWNSGAL(FGly')TP
SRGVHTFP (SEQ ID NO://).

Modification sites As described above, the amino acid sequence of the anti-CD22 antibody is modified in vivo (e.g., in cells by a
during translation of ld-tagged proteins) or in vitro (e.g., in cell-free systems)
FGly by the action of FGE (by contacting the d-tag containing protein with FGE).
It is modified to contain a sulfatase motif containing a serine or cysteine residue that can be converted (oxidized) to a residue. Anti-C used to make conjugates of the present disclosure
The D22 polypeptide comprises at least an Ig constant region, such as an Ig heavy chain constant region (e.g.
at least CH1 domains; at least CH1 and CH2 domains; CH1, CH2 and CH3 domains; or CH1, CH2, CH3 and CH4 domains), or I
g contains the light chain constant region. Such Ig polypeptides are referred to herein as "targeting Ig polypeptides" or "targeting anti-CD22 antibodies" or "targeting anti-CD22 Ig polypeptides".

The site within the anti-CD22 antibody where the sulfatase motif is introduced can be any convenient site. As noted above, the degree of modification of the native amino acid sequence of the target anti-CD22 polypeptide is to minimize the number of amino acid residues inserted, deleted, substituted and/or added (e.g., to the N- or C-terminus). is minimized to Minimizing the degree of amino acid sequence modification of the target anti-CD22 polypeptide can minimize the effects that such modifications can have on anti-CD22 function and/or structure.

The anti-CD22 antibody heavy chain constant region can be of any heavy chain isotype, non-natural Ig heavy chain constant region (
(including consensus Ig heavy chain constant regions). An Ig constant region can be modified to contain an aldehyde tag, where the aldehyde tag is an Ig
Within or adjacent to the solvent accessible loop region of the constant region. To obtain the above sulfatase motif amino acid sequence, the Ig constant regions are
It may be modified by insertion and/or substitution of 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids or more than 16 amino acids.

In some cases, the aldehyde-tagged anti-CD22 antibody is an aldehyde-tagged I
g heavy chain constant region (e.g., at least CH1 domain; at least CH1 and CH2 domains; CH1, CH2 and CH3 domains; or CH1, CH2, CH3 and C
H4 domain). Aldehyde-tagged Ig heavy chain constant regions are IgA, IgM, IgD, IgE, IgG1, IgG2, IgA, IgM, IgD, IgE, IgG1, IgG2, Ig modified to contain at least one sulfatase motif that can be modified by FGE to obtain an FGly-modified Ig polypeptide.
heavy chain constant region sequences of a gG3 or IgG4 isotype heavy chain or any allotypic variant thereof, e.g., human heavy chain constant region sequences or monkey heavy chain constant region sequences, hybrid heavy chain constant regions, synthetic heavy chain constant regions, or consensus It may include heavy chain constant region sequences and the like. Allotypic variants of Ig heavy chains are known in the art. See, eg, Jefferis and Lefranc (2009) MAbs 1:4.

In some cases, the aldehyde-tagged anti-CD22 antibody is an aldehyde-tagged I
g contains the light chain constant region. aldehyde-labeled Ig light chain constant region sequences of kappa light chain, lambda light chain constant region modified to contain at least one sulfatase motif that can be modified by FGE to obtain an FGly-modified anti-CD22 antibody polypeptide; For example, it may include human kappa or lambda light chain constant regions, hybrid light chain constant regions, synthetic light chain constant regions or consensus light chain constant region sequences, and the like. Exemplary constant regions include human gamma 1
and gamma 3 regions. Except for the sulfatase motif, the modified constant region may have a wild-type amino acid sequence, or it is at least 70% identical (e.g., at least 80%, at least 90%, or at least 95% identical) to the wild-type amino acid sequence. ).

In some embodiments, the sulfatase motif is present at a position other than or in addition to the C-terminus of the Ig polypeptide heavy chain. As described above, the isolated aldehyde-tagged anti-CD22 polypeptide can comprise a heavy chain constant region modified to contain a sulfatase motif as described above, wherein the sulfatase motif is an anti-CD22 poly Located at or adjacent to the surface accessible loop region of the peptide heavy chain constant region.

In some cases, the targeted anti-CD22 immunoglobulin is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues. 2) amino acids 137-143; 3) amino acids 155-158; 4) amino acids 163-170; 5) amino acids 163-183; 183; 7
) amino acids 190-192; 8) amino acids 200-202; 9) amino acids 199-202
10) amino acids 208-212; 11) amino acids 220-241; 12) amino acids 24
13) amino acids 257-261; 14) amino acids 269-277; 15) amino acids 271-277; 16) amino acids 284-285; 17) amino acids 284-292
18) amino acids 289-291; 19) amino acids 299-303; 20) amino acids 30
21) amino acids 320-322; 22) amino acids 329-335; 23) amino acids 341-349; 24) amino acids 342-348; 25) amino acids 356-365
26) amino acids 377-381; 27) amino acids 388-394; 28) amino acids 39
8-407; 29) amino acids 433-451; and 30) amino acids 446-451.
Within or adjacent to the region of the IgG1 heavy chain constant region corresponding to the above, wherein the amino acid numbering is based on that of human IgG as shown in Figure 9B.

In some cases, the targeted anti-CD22 immunoglobulin is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues. In some embodiments, the sulfatase motif is 1) amino acids 1-6; 2) amino acids 16-22; 3) amino acids 34-47; 4) amino acids 42-49
5) amino acids 42-62; 6) amino acids 34-37; 7) amino acids 69-71; 8) amino acids 79-81; 9) amino acids 78-81; 12) amino acids 127-131; 13) amino acids 137-141; 1
4) amino acids 149-157; 15) amino acids 151-157; 16) amino acids 164-
165; 17) amino acids 164-172; 18) amino acids 169-171; 19) amino acids 179-183; 20) amino acids 189-193; 21) amino acids 200-202;
2) amino acids 209-215; 23) amino acids 221-229; 24) amino acids 22-2
28; 25) amino acids 236-245; 26) amino acids 217-261; 27) amino acids 268-274; 28) amino acids 278-287; Located within or adjacent to a region of the IgG1 heavy chain constant region, wherein the amino acid numbering is SEQ ID NO // (human IgG1
based on the amino acid numbering of human IgG1 as described in the constant region; sequence shown in FIG. 9B).

Exemplary surface-accessible loop regions of an IgG1 heavy chain include: 1) ASTKGP (SEQ ID NO: //); 2) KSTSGGT (SEQ ID NO: //), shown in Figures 9A and 9B. 3) PEPV (SEQ ID NO //); 4) NSGALTSG (SEQ ID NO //)
5) NSGALTSGVHTFPAVLQSSGL (SEQ ID NO://); 6) QSSGL
(SEQ ID NO//); 7) VTV; 8) QTY; 9) TQTY (SEQ ID NO//); 10) H
KPSN (SEQ ID NO//); 11) EPKSCDKTHTCPPCPAPELLGG (SEQ ID NO//); 12) FPPKP (SEQ ID NO//); 13) ISRTP (SEQ ID NO//)
14) DVSHEDPEV (SEQ ID NO //); 15) SHEDPEV (SEQ ID NO //)
16) DG; 17) DGVEVHNAK (SEQ ID NO: //); 18) HNA; 19) QY
NST (SEQ ID NO//); 20) VLTVL (SEQ ID NO//); 21) GKE; 22) N
KALPAP (SEQ ID NO//); 23) SKAKGQPRE (SEQ ID NO//); 24) K
AKGQPR (SEQ ID NO //); 25) PPSRKELTKN (SEQ ID NO //); 26)
YPSDI (SEQ ID NO //); 27) NGQPENN (SEQ ID NO //); 28) TPPV
LDSDGS (SEQ ID NO//); 29) HEALHNHYTQKSLSLSPGK (SEQ ID NO//); and 30) SLSPGK (SEQ ID NO//).

In some cases, the target immunoglobulin is modified to contain a sulfatase motif as described above, wherein the modification involves insertion, deletion and/or substitution of one or more amino acid residues. In certain embodiments, the sulfatase motif comprises 1) amino acids 1-6;
2) amino acids 13-24; 3) amino acids 33-37; 4) amino acids 43-54; 5) amino acids 58-63; 6) amino acids 69-71;
9) amino acids 95-96; 10) 114-118; 11) 122-126; 12) 134
-136; 13) 144-152; 14) 159-167; 15) 175-176;
) 184-188; 17) 195-197; 18) 204-210; 19) 216-22
4; 20) 231-233; 21) 237-241; 22) 252-256; 23) 26
24) 273-282; 25) within or adjacent to a region of the IgG2 heavy chain constant region corresponding to one or more of amino acids 299-302, wherein the amino acid numbering is SEQ ID NO: // (human IgG2 constant region; also shown in Figure 9B).

Exemplary surface-accessible loop regions of an IgG2 heavy chain include: 1) ASTKGP (SEQ ID NO: //); 2) PCSRSTSESTAA (SEQ ID NO: //); ) FPEPV (SEQ ID NO //); 4) SGALTSGVHTFP (SEQ ID NO //); 5) QSSGLY (SEQ ID NO //); 6) VTV; 7) TQT;
9) DK; 10) VAGPS (SEQ ID NO//); 11) FPPKP (SEQ ID NO//);
12) RTP; 13) DVSHEDPEV (SEQ ID NO: //); 14) DGVEVHNAK
(SEQ ID NO //); 15) FN; 16) VLTVV (SEQ ID NO //); 17) GKE;
8) NKGLPAP (SEQ ID NO//); 19) SKTKGQPRE (SEQ ID NO//); 2
0) PPS; 21) MTKNQ (SEQ ID NO://); 22) YPSDI (SEQ ID NO://);
23) NGQPENN (SEQ ID NO //); 24) TPPMLDSDGS (SEQ ID NO //)
25) GNVF (SEQ ID NO: //); and 26) HEALHNHYTQKSLSLSP
GK (SEQ ID NO://).

In some cases, the target immunoglobulin is modified to contain a sulfatase motif as described above, wherein the modification involves insertion, deletion and/or substitution of one or more amino acid residues. In certain embodiments, the sulfatase motif comprises 1) amino acids 1-6;
2) amino acids 13-22; 3) amino acids 33-37; 4) amino acids 43-61; 5) amino acids 71; 6) amino acids 78-80;
111-115; 10) 147-167; 11) 173-177; 16) 185-187
13) 195-203; 14) 210-218; 15) 226-227; 16) 238
-239; 17) 246-248; 18) 255-261; 19) 267-275;
) 282-291; 21) amino acids 303-307; 22) amino acids 313-320;
3) amino acids 324-333; 24) amino acids 350-352; 25) amino acids 359-
365; and 26) within or adjacent to the region of the IgG3 heavy chain constant region corresponding to one or more of amino acids 372-377, wherein the amino acid numbering is SEQ ID NO://(
human IgG3 constant region; also shown in FIG. 9B).

Exemplary surface-accessible loop regions of an IgG3 heavy chain include: 1) ASTKGP (SEQ ID NO: //); 2) PCSRSTSGGT (SEQ ID NO: //); ) FPEPV (SEQ ID NO //); 4) SGALTSGVHTFPAVLQS
SG (SEQ ID NO//); 5) V; 6) TQT; 7) HKPSN (SEQ ID NO//); 8) R
VELKTPLGD (SEQ ID NO//); 9) CPRCPKP (SEQ ID NO//); 10) P
KSCDTPPPPCPRCPAPELLGG (SEQ ID NO//); 11) FPPKP (SEQ ID NO//); 12) RTP; 13) DVSHEDPEV (SEQ ID NO//); 14) DGV
EVHNAK (SEQ ID NO://); 15) YN; 16) VL; 17) GKE; 18) NKA
LPAP (SEQ ID NO//); 19) SKTKGQPRE (SEQ ID NO//); 20) PPS
REEMTKN (SEQ ID NO //); 21) YPSDI (SEQ ID NO //); 22) SSGQ
PENN (SEQ ID NO //); 23) TPPMLDSDGS (SEQ ID NO //); 24) GN
I; 25) HEALHNR (SEQ ID NO//); and 26) SLSPGK (SEQ ID NO//
).

In some cases, the target immunoglobulin is modified to contain a sulfatase motif as described above, wherein the modification involves insertion, deletion and/or substitution of one or more amino acid residues. In some embodiments, the sulfatase motif comprises 1) amino acids 1-5;
2) amino acids 12-23; 3) amino acids 32-36; 4) amino acids 42-53; 5) amino acids 57-62; 6) amino acids 68-70;
-88; 9) amino acids 94-95; 10) amino acids 101-102; 11) amino acids 10
12) amino acids 122-126; 13) amino acids 134-136; 14) amino acids 144-152; 15) amino acids 159-167; 16) amino acids 175-176
17) amino acids 185-186; 18) amino acids 196-198; 19) amino acids 20
20) amino acids 217-226; 21) amino acids 232-241; 22) amino acids 253-257; 23) amino acids 264-265; 24) 269-270;
26) amino acids 300-303; 27) amino acids 399-41
7, wherein the amino acid numbering is SEQ ID NO://(human IgG4 constant region; also shown in FIG. 9B). based on the numbering of the amino acid sequences described in

Exemplary surface-accessible loop regions of an IgG4 heavy chain include: 1) STKGP (SEQ ID NO: //); 2) PCSRSTSESTAA (SEQ ID NO: //); ) FPEPV (SEQ ID NO //); 4) SGALTSGVHTFP (SEQ ID NO //); 5) QSSGLY (SEQ ID NO //); 6) VTV; 7) TKT;
9) DK; 10) YG; 11) CPAPEFLGGPS (SEQ ID NO: //); 12) FPP
KP (SEQ ID NO//); 13) RTP; 14) DVSQEDPEV (SEQ ID NO//); 1
16) FN; 17) VL; 18) GKE; 1
9) NKGLPSS (SEQ ID NO//); 20) SKAKGQPREP (SEQ ID NO//);
21) PPSQEEMTKN (SEQ ID NO //); 22) YPSDI (SEQ ID NO //); 2
3) NG; 24) NN; 25) TPPVLDSDGS (SEQ ID NO: //); 26) GNVF
(SEQ ID NO//); and 27) HEALHNHYTQKSLSLSLGK (SEQ ID NO//
/).

In some cases, the target immunoglobulin is modified to contain a sulfatase motif as described above, wherein the modification involves insertion, deletion and/or substitution of one or more amino acid residues. In certain embodiments, the sulfatase motif comprises 1) amino acids 1-13
2) amino acids 17-21; 3) amino acids 28-32; 4) amino acids 44-54; 5) amino acids 60-66; 6) amino acids 73-76;
9) amino acids 123-125; 10) amino acids 130-133; 11) amino acids 138-142; 12) amino acids 151-158; 13) amino acids 165-174;
4) amino acids 181-184; 15) amino acids 192-195; 16) amino acids 199;
17) amino acids 209-210; 18) amino acids 222-245; 19) amino acids 252
20) amino acids 266-276; 21) amino acids 293-294; 22) amino acids 301-304; 23) amino acids 317-320; 24) amino acids 329-353. within or adjacent to it, wherein amino acid numbering is based on the numbering of the amino acid sequence set forth in SEQ ID NO: // (human IgA; also shown in Figure 9B).

Exemplary surface accessible loop regions of the IgA heavy chain include: 1) ASPTSPKVFPLSL (SEQ ID NO://); 2) QPDGN (SEQ ID NO://); ) VQGFFPQEPL (SEQ ID NO://); 4) SGQGVTARNFP
(SEQ ID NO//); 5) SGDLYTT (SEQ ID NO//); 6) PATQ (SEQ ID NO//
); 7) GKS; 8) YT; 9) CHP; 10) HRPA (SEQ ID NO: //); 11) LL
GSE (SEQ ID NO //); 12) GLRDASGV (SEQ ID NO //); 13) SSGKS
14) GCYS (SEQ ID NO//); 15) CAEP (SEQ ID NO//); 16) PE; 17) SGNTFRPEVHLLPPPSEELALNEL (
18) ARGFS (SEQ ID NO//); 19) QGSQELPREKY (
20) AV; 21) AAED (SEQ ID NO//); 22) HEAL (SEQ ID NO//); and 23) IDRLAGKPTHVNVSVVMAEVDGTCY (SEQ ID NO//).

Sulfatase motifs may be provided within or adjacent to these amino acid sequences of such modification sites of the Ig heavy chain. For example, the flanking N-terminal side of these modification sites and/or
Alternatively, an Ig heavy chain polypeptide may be modified in one or more of these amino acid sequences to provide a sulfatase motif adjacent to the C-terminus (e.g., where the modification is the insertion, deletion, or deletion of one or more amino acid residues). (including loss and/or replacement). Alternatively or additionally, I
An Ig heavy chain polypeptide may be modified in one or more of these amino acid sequences to provide a sulfatase motif between any two residues of the g heavy chain modification site (e.g., where the modifications are insertions, deletions and/or substitutions of one or more amino acid residues). In some embodiments, the Ig heavy chain polypeptide can be modified to contain two motifs, which may be adjacent to each other, or one, two, three
, may be separated by 4 or more (eg, about 1 to about 25, about 25 to about 50, or about 50 to about 100, or more) amino acids. Alternatively or additionally, modifying selected amino acid residues at the site of modification of the Ig heavy chain polypeptide amino acid sequence, where the native amino acid sequence provides one or more of the amino acid residues of the sulfatase motif sequence, said modification It is possible to obtain sulfatase motifs at sites (e.g.
Here, said modification includes insertion, deletion and/or substitution of one or more amino acid residues).

Thus, the amino acid sequence of the surface-accessible loop region can be modified to obtain a sulfatase motif, where the modifications can include insertions, deletions and/or substitutions. For example, if the modification is in the CH1 domain, the surface accessible loop region can have the amino acid sequence NSGALTSG (SEQ ID NO://) and the aldehyde tagged sequence is, for example, NSGALCTPSRG (SEQ ID NO://). For example, where the "TS" residues of the NSGALTSG (SEQ ID NO://) sequence are "CT
PSR" (SEQ ID NO://) and the sulfatase motif has the sequence LCTPS
R (SEQ ID NO://). As another example, if the modification is in the CH2 domain, the surface-accessible loop region can have the amino acid sequence NKALPAP (SEQ ID NO://) and the aldehyde-tagged sequence is, for example, NLCTPSRAP (sequence #//), for example, where the “KAL” residue of the NKALPAP (SEQ ID NO://) sequence is replaced with “LCTPSR” (SEQ ID NO://) and the sulfatase motif is It has the sequence LCTPSR (SEQ ID NO://). As another example, the surface accessible loop region has the amino acid sequence K when the modification is in the CH2/CH3 main.
AKGQPR (SEQ ID NO://) and the aldehyde-tagged sequence can be for example KAKGLCTPSR (SEQ ID NO://), for example where:
The "GQP" residues of the KAKGQPR (SEQ ID NO//) sequence are replaced with "LCTPS" (SEQ ID NO//
) and the sulfatase motif has the sequence LCTPSR (SEQ ID NO: //).

As described above, an isolated aldehyde-tagged anti-CD22 Ig polypeptide can comprise a light chain constant region modified to contain a sulfatase motif as described above, wherein the sulfatase motif is an Ig poly Within or adjacent to the surface accessible loop region of the peptide light chain constant region. Illustrative representatives of surface accessible loop regions of light chain constant regions are shown in Figures 9A and 9C.

In some cases, the target immunoglobulin is modified to contain a sulfatase motif as described above, wherein the modification involves insertion, deletion and/or substitution of one or more amino acid residues. In certain embodiments, the sulfatase motif comprises: 1) amino acids 130-
135; 2) amino acids 141-143; 3) amino acids 150; 4) amino acids 162-16
6; 5) amino acids 163-166; 6) amino acids 173-180; 7) amino acids 186-
194; 8) amino acids 211-212; 9) amino acids 220-225; 10) amino acids 2
33-236, wherein the amino acid numbering is that of the human kappa light chain amino acid sequence shown in FIG. 9C. based on attachment. In some cases, the target immunoglobulin is modified to contain a sulfatase motif as described above, wherein the modification involves insertion, deletion and/or substitution of one or more amino acid residues. In some embodiments, the sulfatase motif is 1
) amino acids 1-6; 2) amino acids 12-14; 3) amino acids 21; 4) amino acids 33-3.
7) amino acids 34-37; 6) amino acids 44-51; 7) amino acids 57-65; 8)
9) amino acids 91-96; 10) amino acids 104-107 within or adjacent to a region of the Ig light chain constant region corresponding to one or more of amino acids 83-83; Based on number // (human kappa light chain; amino acid sequence set forth in Figure 9C).

Exemplary surface-accessible loop regions of Ig light chains (eg, human kappa light chains) include: 1) RTVAAP (SEQ ID NO://), shown in Figures 9A and 9C;
2) PPS; 3) Gly (see, for example, Gly at position 150 of the human kappa light chain sequence shown in Figure 9C); 4) YPREA (SEQ ID NO: //); 5) PREA (SEQ ID NO: // ); 6) DNALQSGN (SEQ ID NO//); 7) TEQDSKDST (SEQ ID NO//
/); 8) HK; 9) HQGLSS (SEQ ID NO://); and 10) RGEC (SEQ ID NO://).

Exemplary surface accessible loop regions of Ig lambda light chains include: Figure 9C
QPKAAP (SEQ ID NO://), PPS, NK, DFYPGAV (SEQ ID NO://), DSSPVKAG (SEQ ID NO://), TTP, SN, HKS, EG and A
PTECS (SEQ ID NO://).

In some cases, the target immunoglobulin is modified to contain a sulfatase motif as described above, wherein the modification involves insertion, deletion and/or substitution of one or more amino acid residues. In certain embodiments, the sulfatase motif comprises 1) amino acids 1-6;
2) amino acids 12-14; 3) amino acids 121-22; 4) amino acids 31-37; 5) amino acids 44-51; 6) amino acids 55-57;
1-83; 9) amino acids 91-92; 10) amino acids 102-105, wherein the amino acid numbering is: Based on the amino acid sequence numbering of the rat light chain shown in SEQ ID NO// (sequence shown in Figure 9C).

In some cases, the sulfatase motif is CH1 of the anti-CD22 heavy chain constant region.
introduced into the region. In some cases, the sulfatase motif is introduced at or near the C-terminus of the anti-CD22 heavy chain (eg, within 1-10 amino acids of the C-terminus). In some cases, a sulfatase motif is introduced into the light chain constant region.

In some cases, the sulfatase motif is CH1 of the anti-CD22 heavy chain constant region.
within the region, eg, amino acids 121-2 of the IgG1 heavy chain amino acid sequence shown in FIG. 9A
Introduced within 19. For example, in some cases, the sulfatase motif has the amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDKKVE (SEQ ID NO://).
For example, in some of these embodiments, the amino acid sequence GALTSGVH (SEQ ID NO://) is modified to GALCTPSRGVH (SEQ ID NO://), where the sulfatase motif is LCTPSR (SEQ ID NO://).

In some cases, the sulfatase motif is introduced at or near the C-terminus of the anti-CD22 heavy chain, e.g., the sulfatase motif is 1 amino acid, 2 amino acids (aa), 3 aa, 4 aa, 5aa, 6aa, 7aa, 8aa, 9a
a or within 10 aa. As one non-limiting example, the C-terminal lysine residue of the anti-CD22 heavy chain can be replaced with the amino acid sequence SLCTPSRGS (SEQ ID NO://).

In some cases, a sulfatase motif is introduced into the constant region of the light chain of the anti-CD22 antibody. In one non-limiting example, in some cases a sulfatase motif is introduced into the constant region of the light chain of an anti-CD22 antibody, where the sulfatase motif is C-terminal to KVDNAL (SEQ ID NO: //) side and/or QSGNSQ (SEQ ID NO/
/) on the N-terminal side. For example, in some cases the sulfatase motif is LCTPSR (SEQ ID NO://) and the anti-CD22 light chain is the amino acid sequence KVDALL
CTPSRQSGNSQ (SEQ ID NO://).

Exemplary Anti-CD22 Antibodies In some cases, a suitable anti-CD22 antibody may be a CD22 epitope (eg, within amino acids 1-847, within amino acids 1-847 of the CD22 amino acid sequences shown in FIGS. 8A-8C).
759, within amino acids 1-751 or within amino acids 1-670), IYDMS (VH CDR1; SEQ ID NO://), YISSGGGTTYYPDT
VKG (VH CDR2; SEQ ID NO: //) and HSGYGSSYGVLFAY (VH
CDR3; In some cases, the anti-CD22 antibody is humanized. In some cases
The anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues. In some embodiments, the sulfatase motif is 1) amino acids 1-6; 2) amino acids 16-22; 3)
4) amino acids 42-49; 5) amino acids 42-62; 6) amino acids 34-37; 7) amino acids 69-71; 8) amino acids 79-81;
1; 10) amino acids 87-91; 11) amino acids 100-121; 12) amino acids 127
13) amino acids 137-141; 14) amino acids 149-157; 15) amino acids 151-157; 16) amino acids 164-165; 17) amino acids 164-172;
18) amino acids 169-171; 19) amino acids 179-183; 20) amino acids 189
21) amino acids 200-202; 22) amino acids 209-215; 23) amino acids 221-229; 24) amino acids 22-228; 25) amino acids 236-245;
6) amino acids 217-261; 27) amino acids 268-274; 28) amino acids 278-
287; 29) amino acids 313-331; and 30) amino acids 324-331, wherein the amino acid numbering is SEQ ID NO: Based on the amino acid numbering of human IgG1 shown in // (human IgG1 constant region shown in Figure 9B). In some cases, the anti-CD
22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues, e.g., wherein the sulfatase motif comprises: 1) an amino acid 1-6; 2) amino acids 12-14; 3) amino acids 21; 4
6) amino acids 44-51; 7) amino acids 57-65; 8) amino acids 83-83; 9) amino acids 91-96;
4-107, wherein the amino acid numbering is SEQ ID NO://(human kappa light chain; amino acids shown in FIG. 9C) array).

In some cases, a suitable anti-CD22 antibody includes a CD22 epitope (eg, within amino acids 1-847, amino acids 1-847 of the CD22 amino acid sequences shown in FIGS. 8A-8C).
759, within amino acids 1-751 or within amino acids 1-670), RASQDISNYLN (VL CDR1;
VL CDR2; SEQ ID NO//) and QQGNTLPWT (VL CDR3; SEQ ID NO//). In some cases, the anti-CD22 antibody is humanized. In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues. In certain embodiments, the sulfatase motif is 1) amino acids 122-127; 2) amino acids 137-143; 3) amino acid 15
5) amino acids 163-183; 6) amino acids 179-183; 7) amino acids 190-192; 8) amino acids 200-202; 9) amino acids 199-202; -212; 11) amino acids 220-241;
12) amino acids 247-251; 13) amino acids 257-261; 14) amino acids 269
15) amino acids 271-277; 16) amino acids 284-285; 17) amino acids 284-292; 18) amino acids 289-291; 19) amino acids 299-303;
20) amino acids 309-313; 21) amino acids 320-322; 22) amino acids 329
23) amino acids 341-349; 24) amino acids 342-348; 25) amino acids 356-365; 26) amino acids 377-381; 27) amino acids 388-394;
28) amino acids 398-407; 29) amino acids 433-451; and 30) amino acids 446-451, wherein the amino acid number The numbering is based on the human IgG1 numbering shown in Figure 9B. In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues, e.g. and the sulfatase motif consists of 1) amino acids 1-6;
) amino acids 12-14; 3) amino acids 21; 4) amino acids 33-37; 5) amino acids 34
-37; 6) amino acids 44-51; 7) amino acids 57-65; 8) amino acids 83-83;
9) within or adjacent to a region of the Ig kappa constant region corresponding to one or more of amino acids 91-96; 10) amino acids 104-107, wherein the amino acid numbering is SEQ ID NO // (human kappa chain; amino acid sequence shown in FIG. 9C).

In some cases, a suitable anti-CD22 antibody includes a CD22 epitope (eg, within amino acids 1-847, amino acids 1-847 of the CD22 amino acid sequences shown in FIGS. 8A-8C).
759, within amino acids 1-751 or within amino acids 1-670), VH CDRs: IYDMS (VH CDR1; SEQ ID NO//), YISSGGG
TTYYPDTVKG (VH CDR2; SEQ ID NO://) and HSGYGSSYGVL
Compete with antibodies containing FAY (VH CDR3; SEQ ID NO: //). In some cases, the anti-CD22 antibody is humanized. In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues. In some embodiments, the sulfatase motif is 1) amino acids 1-6; 2) amino acids 16-22; 3) amino acids 34-4.
7; 4) amino acids 42-49; 5) amino acids 42-62; 6) amino acids 34-37;
8) amino acids 79-81; 9) amino acids 78-81; 10) amino acids 87-91; 11) amino acids 100-121; 12) amino acids 127-131;
14) amino acids 149-157; 15) amino acids 151-15
7; 16) amino acids 164-165; 17) amino acids 164-172; 18) amino acids 1
69-171; 19) amino acids 179-183; 20) amino acids 189-193; 21)
22) amino acids 209-215; 23) amino acids 221-22
9; 24) amino acids 22-228; 25) amino acids 236-245; 26) amino acids 21
27) amino acids 268-274; 28) amino acids 278-287; 29) amino acids 313-331; and 30) amino acids 324-331.
Within or adjacent to a region of a single heavy chain constant region, wherein the amino acid numbering is
human IgG shown in SEQ ID NO: // (human IgG1 constant region shown in Figure 9B)
Based on 1 amino acid numbering. In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues, e.g. and the sulfatase motif is
1) amino acids 1-6; 2) amino acids 12-14; 3) amino acids 21; 4) amino acids 33-
37; 5) amino acids 34-37; 6) amino acids 44-51; 7) amino acids 57-65;
9) amino acids 91-96; 10) amino acids 104-107, wherein the amino acid numbering is sequence Based on number // (human kappa light chain; amino acid sequence shown in Figure 9C).

In some cases, a suitable anti-CD22 antibody includes a CD22 epitope (eg, within amino acids 1-847, amino acids 1-847 of the CD22 amino acid sequences shown in FIGS. 8A-8C).
759, within amino acids 1-751 or within amino acids 1-670), VL CDR: RASQDISNYLN (VL CDR1; SEQ ID NO://), Y
TSILHS (VL CDR2; SEQ ID NO://) and QQGNTLPWT (VL CD
R3; SEQ ID NO//). In some cases, the anti-CD22 antibody is humanized. In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues. In some embodiments, the sulfatase motif is 1
) amino acids 1-6; 2) amino acids 16-22; 3) amino acids 34-47; 4) amino acids 4
2-49; 5) amino acids 42-62; 6) amino acids 34-37; 7) amino acids 69-71
8) amino acids 79-81; 9) amino acids 78-81; 10) amino acids 87-91;
) amino acids 100-121; 12) amino acids 127-131; 13) amino acids 137-1
14) amino acids 149-157; 15) amino acids 151-157; 16) amino acids 164-165; 17) amino acids 164-172; 18) amino acids 169-171;
) amino acids 179-183; 20) amino acids 189-193; 21) amino acids 200-2
22) amino acids 209-215; 23) amino acids 221-229; 24) amino acids 22-228; 25) amino acids 236-245; 26) amino acids 217-261;
28) amino acids 278-287; 29) amino acids 313-33
1; and 30) within or adjacent to a region of the IgG1 heavy chain constant region corresponding to one or more of amino acids 324-331, where the amino acid numbering is SEQ ID NO // (FIG. 9)
Based on the amino acid numbering of human IgG1 shown in the human IgG1 constant region shown in B). In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues, e.g. and the sulfatase motif consists of 1) amino acids 1-6
2) amino acids 12-14; 3) amino acids 21; 4) amino acids 33-37; 5) amino acids 34-37; 6) amino acids 44-51;
3; 9) amino acids 91-96; 10) amino acids 104-107 within or adjacent to a region of the Ig kappa constant region corresponding to one or more of the amino acids, wherein the amino acid numbering is SEQ ID NO // ( based on the human kappa light chain; amino acid sequence shown in FIG. 9C).

In some cases, a suitable anti-CD22 antibody includes a CD22 epitope (eg, within amino acids 1-847, amino acids 1-847 of the CD22 amino acid sequences shown in FIGS. 8A-8C).
759, within amino acids 1-751 or within amino acids 1-670), VH CDRs: IYDMS (VH CDR1; SEQ ID NO//), YISSGGG
TTYYPDTVKG (VH CDR2; SEQ ID NO://) and HSGYGSSYGVL
FAY (VH CDR3; SEQ ID NO://) and VL CDR: RASQDISNYL
N (VL CDR1; SEQ ID NO//), YTSILHS (VL CDR2; SEQ ID NO//
) and QQGNTLPWT (VL CDR3; SEQ ID NO://).
In some cases, the anti-CD22 antibody is humanized. In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein
The modifications include insertions, deletions and/or substitutions of one or more amino acid residues. In some embodiments, the sulfatase motif is 1) amino acids 1-6; 2) amino acids 16-22;
3) amino acids 34-47; 4) amino acids 42-49; 5) amino acids 42-62; 6) amino acids 34-37; 7) amino acids 69-71;
-81; 10) amino acids 87-91; 11) amino acids 100-121; 12) amino acids 1
13) amino acids 137-141; 14) amino acids 149-157; 15)
16) amino acids 164-165; 17) amino acids 164-17
2; 18) amino acids 169-171; 19) amino acids 179-183; 20) amino acids 1
21) amino acids 200-202; 22) amino acids 209-215; 23)
24) amino acids 22-228; 25) amino acids 236-245
26) amino acids 217-261; 27) amino acids 268-274; 28) amino acids 27
8-287; 29) amino acids 313-331; and 30) amino acids 324-331.
within or adjacent to the region of the IgG1 heavy chain constant region corresponding to the above, wherein the amino acid numbering is shown in SEQ ID NO: // (human IgG1 constant region shown in Figure 9B) Based on the amino acid numbering of human IgG1 in In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues, e.g. and the sulfatase motif is 1) amino acids 1-6; 2) amino acids 12-14; 3) amino acid 21
4) amino acids 33-37; 5) amino acids 34-37; 6) amino acids 44-51; 7) amino acids 57-65; 8) amino acids 83-83; within or adjacent to a region of the Ig kappa constant region corresponding to one or more, wherein amino acid numbering is based on SEQ ID NO: // (human kappa light chain; amino acid sequence shown in Figure 9C) .

In some cases, a suitable anti-CD22 antibody has the VH CDR: IYDMS (VH
CDR1; SEQ ID NO//), YISSGGGTTYYPDTVKG (VH CDR2; SEQ ID NO//) and HSGYGSSYGVLFAY (VH CDR3; SEQ ID NO//). In some cases, the anti-CD22 antibody is humanized. In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues. In some embodiments, the sulfatase motif is 1) amino acids 1-6; 2) amino acids 16-
22; 3) amino acids 34-47; 4) amino acids 42-49; 5) amino acids 42-62;
7) amino acids 69-71; 8) amino acids 79-81; 9) amino acids 78-81; 10) amino acids 87-91; 11) amino acids 100-121; 14) amino acids 149-157;
15) amino acids 151-157; 16) amino acids 164-165; 17) amino acids 164
18) amino acids 169-171; 19) amino acids 179-183; 20) amino acids 189-193; 21) amino acids 200-202; 22) amino acids 209-215;
23) amino acids 221-229; 24) amino acids 22-228; 25) amino acids 236-
245; 26) amino acids 217-261; 27) amino acids 268-274; 28) amino acids 278-287; 29) amino acids 313-331; and 30) amino acids 324-33
within or adjacent to a region of the IgG1 heavy chain constant region corresponding to one or more of 1, wherein the amino acid numbering is SEQ ID NO // (human IgG1 constant region shown in Figure 9B) based on the amino acid numbering of human IgG1 shown in . In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues, e.g. and,
2) amino acids 12-14; 3) amino acids 21; 4) amino acids 33-37; 5) amino acids 34-37; 6) amino acids 44-51;
7) amino acids 57-65; 8) amino acids 83-83; 9) amino acids 91-96; , amino acid numbering is based on SEQ ID NO: // (human kappa light chain; amino acid sequence shown in Figure 9C).

In some cases, a suitable anti-CD22 antibody has the VL CDR: RASQDISNY
LN (VL CDR1; SEQ ID NO//), YTSILHS (VL CDR2; SEQ ID NO/
/) and QQGNTLPWT (VL CDR3; SEQ ID NO://). In some cases, the anti-CD22 antibody is humanized. In some cases, the anti-CD
22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues. 2) amino acids 16-22; 3) amino acids 34-47; 4) amino acids 42-49; 5) amino acids 42-62; 3
7) amino acids 69-71; 8) amino acids 79-81; 9) amino acids 78-81;
) amino acids 87-91; 11) amino acids 100-121; 12) amino acids 127-131
13) amino acids 137-141; 14) amino acids 149-157; 15) amino acids 15
16) amino acids 164-165; 17) amino acids 164-172; 18) amino acids 169-171; 19) amino acids 179-183; 20) amino acids 189-193
21) amino acids 200-202; 22) amino acids 209-215; 23) amino acids 22
24) amino acids 22-228; 25) amino acids 236-245; 26) amino acids 217-261; 27) amino acids 268-274; 28) amino acids 278-287;
29) within or adjacent to a region of the IgG1 heavy chain constant region corresponding to one or more of amino acids 313-331; and 30) amino acids 324-331, wherein the amino acid numbering is SEQ ID NO// Based on the amino acid numbering of human IgG1 shown in (human IgG1 constant region shown in FIG. 9B). In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues, e.g. 2) amino acids 12-14; 3) amino acids 21; 4) amino acids 33-37; 5) amino acids 34-37; 6) amino acids 44-51; -
65; 8) amino acids 83-83; 9) amino acids 91-96; 10) amino acids 104-10
within or adjacent to the region of the Ig kappa constant region corresponding to one or more of 7, wherein the amino acid numbering is SEQ ID NO: // (human kappa light chain; amino acid sequence shown in Figure 9C) based on.

In some cases, a suitable anti-CD22 antibody has the VH CDR: IYDMS (VH
CDR1; SEQ ID NO//), YISSGGGTTYYPDTVKG (VH CDR2; SEQ ID NO//) and HSGYGSSYGVLFAY (VH CDR3; SEQ ID NO//) and VL CDRs: RASQDISNYLN (VL CDR1; SEQ ID NO//), YT
SILHS (VL CDR2; SEQ ID NO://) and QQGNTLPWT (VL CDR
3; SEQ ID NO//). In some cases, the anti-CD22 antibody is humanized. In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues. In certain embodiments, the sulfatase motif comprises: 1) amino acids 1-6
2) amino acids 16-22; 3) amino acids 34-47; 4) amino acids 42-49; 5) amino acids 42-62; 6) amino acids 34-37;
9) amino acids 78-81; 10) amino acids 87-91; 11) amino acids 100
12) amino acids 127-131; 13) amino acids 137-141; 14) amino acids 149-157; 15) amino acids 151-157; 16) amino acids 164-165;
17) amino acids 164-172; 18) amino acids 169-171; 19) amino acids 179
20) amino acids 189-193; 21) amino acids 200-202; 22) amino acids 209-215; 23) amino acids 221-229; 24) amino acids 22-228;
5) amino acids 236-245; 26) amino acids 217-261; 27) amino acids 268-
274; 28) amino acids 278-287; 29) amino acids 313-331; and 30)
within or adjacent to a region of the IgG1 heavy chain constant region corresponding to one or more of amino acids 324-331, wherein the amino acid numbering is SEQ ID NO // (human IgG1 based on the amino acid numbering of human IgG1 given in the constant region). In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues, e.g. and the sulfatase motif is 1) amino acids 1-6; 2) amino acid 1
2-14; 3) amino acids 21; 4) amino acids 33-37; 5) amino acids 34-37;
7) amino acids 57-65; 8) amino acids 83-83; 9) amino acids 91-96; 10) amino acids 104-107. where amino acid numbering is based on SEQ ID NO: // (human kappa light chain; amino acid sequence shown in Figure 9C).

In some cases, a suitable anti-CD22 antibody has the following amino acid sequence: EVQLV
ESGGGLVKPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGL
EWVAYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMSS
Contains VH CDRs present within the anti-CD22 VH region including LRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS (SEQ ID NO://). In some cases, the anti-CD22 antibody is humanized. In some cases, the anti-C
The D22 antibody is modified to contain a sulfatase motif as described above, wherein the modification is 1
Including insertions, deletions and/or substitutions of the above amino acid residues. In some embodiments,
2) amino acids 16-22; 3) amino acids 34-47; 4) amino acids 42-49; 5) amino acids 42-62;
37; 7) amino acids 69-71; 8) amino acids 79-81; 9) amino acids 78-81;
0) amino acids 87-91; 11) amino acids 100-121; 12) amino acids 127-13
1; 13) amino acids 137-141; 14) amino acids 149-157; 15) amino acid 1
16) amino acids 164-165; 17) amino acids 164-172; 18)
19) amino acids 179-183; 20) amino acids 189-19
3; 21) amino acids 200-202; 22) amino acids 209-215; 23) amino acids 2
21-229; 24) amino acids 22-228; 25) amino acids 236-245; 26) amino acids 217-261; 27) amino acids 268-274;
29) amino acids 313-331; and 30) amino acids 324-331, wherein the amino acid numbering is SEQ ID NO:/ Based on the amino acid numbering of human IgG1 shown in / (human IgG1 constant region shown in Figure 9B). In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues, e.g. 2) amino acids 12-14; 3) amino acids 21; 4) amino acids 33-37; 5) amino acids 34-37; 6) amino acids 44-51;
-65; 8) amino acids 83-83; 9) amino acids 91-96; 10) amino acids 104-1
07, wherein the amino acid numbering is SEQ ID NO:// (human kappa light chain; amino acid sequence shown in FIG. 9C) based on.

In some cases, a suitable anti-CD22 antibody has the following amino acid sequence: DIQMT
QSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVK
LLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQQEDFAT
Anti-CD22 containing YFCQQGNTLPWTFGGGTKVEIKR (SEQ ID NO://)
Contains the VL CDRs that reside within the VL region. In some cases, the anti-CD22 antibody is humanized. In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues. In some embodiments, the sulfatase motif is 1)
2) amino acids 16-22; 3) amino acids 34-47; 4) amino acids 42
-49; 5) amino acids 42-62; 6) amino acids 34-37; 7) amino acids 69-71;
8) amino acids 79-81; 9) amino acids 78-81; 10) amino acids 87-91; 11)
12) amino acids 127-131; 13) amino acids 137-14
1; 14) amino acids 149-157; 15) amino acids 151-157; 16) amino acid 1
17) amino acids 164-172; 18) amino acids 169-171; 19)
20) amino acids 189-193; 21) amino acids 200-20
2; 22) amino acids 209-215; 23) amino acids 221-229; 24) amino acid 2
25) amino acids 236-245; 26) amino acids 217-261; 27) amino acids 268-274; 28) amino acids 278-287; 29) amino acids 313-331
and 30) within or adjacent to the region of the IgG1 heavy chain constant region corresponding to one or more of amino acids 324-331, wherein the amino acid numbering is SEQ ID NO // (Figure 9B
based on the amino acid numbering of human IgG1 shown in (human IgG1 constant region shown in ). In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises the insertion, deletion and/or insertion of one or more amino acid residues.
or substitutions, for example, wherein the sulfatase motif is 1) amino acids 1-6;
2) amino acids 12-14; 3) amino acids 21; 4) amino acids 33-37; 5) amino acids 3
4-37; 6) amino acids 44-51; 7) amino acids 57-65; 8) amino acids 83-83
9) amino acids 91-96; 10) within or adjacent to a region of the Ig kappa constant region corresponding to one or more of amino acids 104-107, wherein the amino acid numbering is SEQ ID NO // (human kappa light chain; amino acid sequence shown in Figure 9C).

In some cases, a suitable anti-CD22 antibody is EVQLVESGGGLVKPG
GSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGG
GTTYYPDTVKGRFTISRDNAKNSLYLQMSSLRAEDTAMYY
VH CDRs present in CARHSGYGSSYGVLFAYWGQGTLVTVSS (SEQ ID NO://) and DIQMTQSPSSLSASVGDRVTITCRASQD
ISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSG
TDYTLTISSLQQEDFATYFCQQGNTLPWTFGGGTKVEIKR
Contains the VL CDRs present in (SEQ ID NO://). In some cases, the anti-CD
22 antibodies have been humanized. In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues. 2) amino acids 16-22; 3) amino acids 34-47; 4) amino acids 42-49; 5) amino acids 42-62; 37; 7) amino acid 69
-71; 8) amino acids 79-81; 9) amino acids 78-81; 10) amino acids 87-91
11) amino acids 100-121; 12) amino acids 127-131; 13) amino acids 13
14) amino acids 149-157; 15) amino acids 151-157; 16) amino acids 164-165; 17) amino acids 164-172; 18) amino acids 169-171
19) amino acids 179-183; 20) amino acids 189-193; 21) amino acids 20
22) amino acids 209-215; 23) amino acids 221-229; 24) amino acids 22-228; 25) amino acids 236-245; 26) amino acids 217-261;
27) amino acids 268-274; 28) amino acids 278-287; 29) amino acids 313
-331; and 30) within or adjacent to the region of the IgG1 heavy chain constant region corresponding to one or more of amino acids 324-331, wherein the amino acid numbering is SEQ ID NO: //
Based on the amino acid numbering of human IgG1 shown in (human IgG1 constant region shown in FIG. 9B). In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues, e.g. 2) amino acids 12-14; 3) amino acids 21; 4) amino acids 33-37; 5) amino acids 34-37; 6) amino acids 44-51; -65; 8) amino acid 8
3-83; 9) amino acids 91-96; 10) amino acids 104-107, wherein the amino acid numbering is SEQ ID NO/ / (human kappa light chain; amino acid sequence shown in Figure 9C).

In some cases, a suitable anti-CD22 antibody has the VH amino acid sequence EVQLVESG
GGLVKPGGSLRRLSCAASGFAFSIYDMSWVRQAPGKGLEWV
AYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMSSLRA
EDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS (SEQ ID NO: //). In some cases, a suitable anti-CD22 antibody has the VL amino acid sequence DI
QMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGK
AVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQQED
FATYFCQQGNTLPWTFGGGTKVEIKR (SEQ ID NO://). In some cases, a suitable anti-CD22 antibody has the VH amino acid sequence EVQLVESGGGL
VKPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYI
SSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMSSLRAEDT
AMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS (SEQ ID NO://)
and VL amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDI
SNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGT
DYTLTISSLQQEDFATYFCQQGNTLPWTFGGGTKVEIKR(
SEQ ID NO //). In some cases, the anti-CD22 antibody is modified to contain a sulfatase motif as described above, wherein the modification comprises insertion, deletion and/or substitution of one or more amino acid residues. In some embodiments, the sulfatase motif is 1
) amino acids 1-6; 2) amino acids 16-22; 3) amino acids 34-47; 4) amino acids 4
2-49; 5) amino acids 42-62; 6) amino acids 34-37; 7) amino acids 69-71
8) amino acids 79-81; 9) amino acids 78-81; 10) amino acids 87-91;
) amino acids 100-121; 12) amino acids 127-131; 13) amino acids 137-1
14) amino acids 149-157; 15) amino acids 151-157; 16) amino acids 164-165; 17) amino acids 164-172; 18) amino acids 169-171;
) amino acids 179-183; 20) amino acids 189-193; 21) amino acids 200-2
22) amino acids 209-215; 23) amino acids 221-229; 24) amino acids 22-228; 25) amino acids 236-245; 26) amino acids 217-261;
28) amino acids 278-287; 29) amino acids 313-33
1; and 30) within or adjacent to a region of the IgG1 heavy chain constant region corresponding to one or more of amino acids 324-331, where the amino acid numbering is SEQ ID NO // (FIG. 9)
Based on the amino acid numbering of human IgG1 shown in the human IgG1 constant region shown in B).

Drugs for Conjugation to Polypeptides The present disclosure provides drug-polypeptide conjugates. Examples of drugs include small molecule drugs,
Examples include cancer chemotherapeutic agents. For example, when the polypeptide is an antibody (or fragment thereof) with specificity for tumor cells, the antibody comprises modified amino acids that are subsequently conjugated to a cancer chemotherapeutic agent such as a microtubule acting agent. As such, it can be modified as described herein. In some embodiments, the drug is a maytansinoid, which has the structure:

As noted above, in some embodiments, L is of the formula -(L ¹ ) _a -(L ² ) _b -(L ³
) _c -(L ⁴ ) _d -, where L ¹ , L ² , L ³ and L
⁴ is each independently a linker unit. In certain embodiments, L ¹ is attached to a coupling moiety such as a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety (eg, those shown in formula (I) above). In some embodiments, L ² , if present, is attached to W ¹ (maytansinoid). In certain embodiments, L ³ , when present, is W ¹ (maytansinoid)
is bound to In certain embodiments, L ⁴ , if present, is attached to W ¹ (maytansinoid).

As noted above, in some embodiments, the linker: -(L ¹ ) _a -(L ² ) _b -(L
³ ) _c -(L ⁴ ) _d - is the formula: -(T ¹ -V ¹ ) _a -(T ² -V ² ) _b -(T ³ -V ³ ) _c
—(T ⁴ —V ⁴ ) _d —, wherein a, b, c and d are each independently 0 or 1 and the sum of a, b, c and d is 1 to 4 is. In certain embodiments, as described above, L ¹ is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety (eg, those shown in formula (I) above).
Thus, in certain embodiments, T ¹ is attached to a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety (eg, those shown in formula (I) above). In some embodiments, V ¹ is attached to W ¹ (maytansinoid). In certain embodiments, L ² , if present, is attached to W ¹ (maytansinoid), as described above. Thus, in certain embodiments, T ² , if present, is bound to W ¹ (maytansinoid), or V ² , if present, is bound to W ¹ (maytansinoid) Combined. In certain embodiments, L ³ , if present, is attached to W ¹ (maytansinoid), as described above. Thus, in certain embodiments, T3 ^, if present, is bound to W1 (maytansinoid), or ^{V3 ,} if present, is bound to ^W1 (maytansinoid) Combined. In certain embodiments, L ⁴ , if present, is attached to W ¹ (maytansinoid). Thus, in certain embodiments, ^T4 , if present, is
bound to W ¹ (maytansinoid) or V ⁴ , if present, W ¹ (
maytansinoids).

Embodiments of the present disclosure provide that the polypeptide (e.g., anti-CD22 antibody) comprises one or more drug moieties (
e.g., maytansinoids), e.g., 2 drug moieties, 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, Includes conjugates that are conjugated to 9 drug moieties or 10 or more drug moieties. A drug moiety can be conjugated to a polypeptide at one or more sites on the polypeptide, as described herein. In some embodiments, the conjugate is 0.1
to 10, or 0.5 to 10, or 1 to 10, such as 1 to 9, or 1 to 8, or 1
have an average drug-to-antibody ratio (DAR) (molar ratio) ranging from ~7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2. In some embodiments, the conjugate is 1-2, such as 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7
, 1.8, 1.9 or 2. In some embodiments, the conjugate has an average DAR of 1.6-1.9. In some embodiments, the conjugate has an average DAR of 1.7. Average means arithmetic mean.

Formulations Conjugates of the disclosure (including antibody conjugates) can be formulated in a variety of ways. Generally, when the conjugate is a polypeptide-drug conjugate, the conjugate is formulated (formulated) in a manner compatible with the drug to which the polypeptide is conjugated, the condition to be treated and the route of administration to be used. be.

The conjugate (eg, polypeptide-drug conjugate) can be in any suitable form,
For example, it can be provided in the form of a pharmaceutically acceptable salt and formulated for any suitable route of administration, such as oral, topical or parenteral. Where the conjugates are provided as liquid injectables (e.g., embodiments in which they are administered intravenously or directly into a tissue), the conjugates may be in a ready-to-use form or reconstituted. It may be provided as a reconstitutable, shelf-stable powder or liquid (consisting of pharmaceutically acceptable carriers and excipients).

Methods for formulating conjugates may be adapted from those readily available. For example, a conjugate can be provided in a pharmaceutical composition comprising a therapeutically effective amount of the conjugate and a pharmaceutically acceptable carrier (eg, saline). The pharmaceutical composition optionally comprises
Other additives (eg, buffers, stabilizers, preservatives, etc.) may be included. In some embodiments, the formulation is suitable for administration to mammals, eg, for administration to humans.

Methods of Treatment Polypeptide-drug conjugates of the disclosure are useful in treating conditions or diseases in subjects amenable to treatment by administration of the parent drug (ie, the drug prior to conjugation to the polypeptide). "Treatment" means that at least amelioration of symptoms associated with the condition afflicting the host is achieved, wherein amelioration is achieved by at least reducing a parameter, e.g., the severity of symptoms, associated with the condition being treated. used in a broad sense to mean Thus, treatment is directed to completely suppressing, e.g., preventing or arresting or terminating the development of, the pathological condition or at least the symptoms associated therewith such that the host develops the condition or at least the symptoms that characterize the condition. Including situations where no haze is present. Therefore, treatment should
(i) prevention of clinical symptoms, i.e., reducing the risk of developing clinical symptoms, e.g., not developing clinical symptoms, e.g., preventing progression of the disease to a detrimental state; , prevention of development or further development of clinical symptoms, eg, alleviation or complete suppression of active disease; and/or (iii) alleviation of clinical symptoms, ie, causing regression of clinical symptoms.

The term "treatment" in the context of cancer refers to any or all of reducing solid tumor growth, inhibiting cancer cell replication, reducing overall tumor burden, and ameliorating one or more symptoms associated with cancer. include.

The subject to be treated can be in need of treatment, wherein the host to be treated can be amenable to treatment with the parent drug. Accordingly, a variety of subjects may be suitable for treatment using the polypeptide-drug conjugates disclosed herein. Generally, such subjects are "mammals" and are of human interest. Other subjects include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs and rats, e.g., in animal models of disease). ) and non-human primates (eg, chimpanzees and monkeys).

The amount of polypeptide-drug conjugate administered will initially be the dose of the parent drug and/or
or can be determined based on dosage regimen guidelines. In general, the polypeptide-drug conjugates are capable of providing targeted delivery and/or improved serum half-life of the bound drug, thus reducing the dose or at least one of reducing administration in the dosing regimen.
can bring about Thus, a Polypeptide-Drug Conjugate can result in a dose reduction and/or administration reduction in an administration regimen as compared to the parent drug prior to conjugation in the Polypeptide-Drug Conjugates of the present disclosure.

Furthermore, as discussed above, since the Polypeptide-Drug Conjugates can provide control of the stoichiometry of drug delivery, the dosage of the Polypeptide-Drug Conjugates can be adjusted to provide per drug molecule per Polypeptide-Drug Conjugate. can be calculated based on the number of

In some embodiments, multiple doses of the polypeptide-drug conjugate are administered. The frequency of administration of the Polypeptide-Drug Conjugate can vary depending on any of a variety of factors such as, eg, severity of symptoms, condition of the subject, and the like. For example, in some embodiments, the polypeptide-drug conjugate is administered once a month, twice a month, three times a month, every other week, once a week (qwk), twice a week, three times a week, four times a week, 5 times a week, 6 times a week, every other day, daily (qd/od), 1
Such as twice daily (bds/bid) or three times daily (tds/tid).

Methods of Treating Cancer The present disclosure provides methods for administering cancer chemotherapeutic agents to individuals with cancer. The methods are useful for treating a wide variety of cancers, including carcinomas, sarcomas, leukemias and lymphomas.

Carcinomas that can be treated using this method include esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), transitional cell carcinoma (a malignant neoplasm of the bladder). bladder cancer, including
lung cancer, including bronchogenic carcinoma, colon cancer, colorectal cancer, gastric cancer, small cell carcinoma and non-small cell carcinoma of the lung;
Adrenal cortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, in situ breast Ductal or cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, uterine cancer, testicular cancer, osteogenic carcinoma, epithelial carcinoma and nasopharyngeal carcinoma, etc. not to be

Sarcoma that can be treated using this method include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synoviosarcoma, include mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma and other soft tissue sarcomas, but
It is not limited to these.

Other solid tumors that can be treated using this method include glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineocytoma, hemangioblastoma, acoustic neuroma, oligodendromas. glioma, meningioma, melanoma,
Including, but not limited to, neuroblastoma and retinoblastoma.

Leukemias that can be treated using this method include: a) chronic myeloproliferative syndrome (a neoplastic disorder of multipotent hematopoietic stem cells); b) acute myeloid leukemia (multipotent hematopoietic stem cells or lineage restricted possible neoplastic transformation of hematopoietic cells); c) chronic lymphocytic leukemia (
CLL; clonal expansion of immunologically immature and dysfunctional small lymphocytes), e.g., B-cell CL
L, T-cell CLL prolymphocytic leukemia and hairy cell leukemia; and d) acute lymphoblastic leukemia (characterized by accumulation of lymphoblasts). Lymphomas that can be treated using this method include B-cell lymphomas (e.g.,
Burkitt's lymphoma), Hodgkin's lymphoma, non-Hodgkin's B-cell lymphoma, and the like, but are not limited to these.

FIG. 1 panel A shows a formylglycine-generating enzyme (FGE) recognition sequence inserted at the desired location along the antibody backbone using standard molecular biology techniques. FGE, which is endogenous to eukaryotic cells, when expressed catalyzes the conversion of Cys within the consensus sequence to a formylglycine residue (FGly). FIG. 1 panel B shows hydrazino-iso-Pictet-Spengler (HIPS) linkers and aldehyde moieties (2 per antibody) reacted with payloads to give site-specific conjugated ADCs. Containing antibodies are shown. FIG. 1 panel C shows HIPS chemistry, which proceeds via an intermediate hydrazonium ion, followed by intramolecular alkylation with a nucleophilic indole to form a stable C—C bond. FIG. 2 shows a hydrophobic interaction column (HIC) trace of an aldehyde-tagged anti-CD22 antibody conjugated at the C-terminus (CT) to a maytansine payload attached to a HIPS-4AP linker according to an embodiment of the present disclosure. . FIG. 3 shows a HIC trace of an aldehyde-tagged anti-CD22 antibody conjugated at the C-terminus (CT) to a maytansine payload attached to a HIPS-4AP linker according to an embodiment of the present disclosure. FIG. 4 shows a reverse phase chromatography (PLRP) trace of an aldehyde-tagged anti-CD22 antibody conjugated at the C-terminus (CT) to a maytansine payload attached to a HIPS-4AP linker according to an embodiment of the present disclosure. FIG. 5 is an analytical size exclusion chromatography (SEC) analysis of an aldehyde-tagged anti-CD22 antibody conjugated at the C-terminus (CT) to a maytansine payload linked to a HIPS-4AP linker according to an embodiment of the present disclosure. Show the graph. FIG. 6A shows in vitro efficacy against WSU-DLCL2 cells for anti-CD22 ADCs conjugated at the C-terminus (CT) to a maytansine payload attached to a HIPS-4AP linker according to embodiments of the present disclosure [Log antibody drug conjugate ( ADC) concentration (nM) versus viability (%)]. FIG. 6B shows in vitro efficacy against Ramos cells for anti-CD22 ADCs conjugated at the C-terminus (CT) to a maytansine payload attached to a HIPS-4AP linker according to embodiments of the present disclosure [Log antibody drug conjugate (ADC) Concentration (nM) vs. Viability (%)]. FIG. 7 depicts in vivo efficacy [mean tumor vs. Volume (mm ³ )]. Figures 8A-8C show the amino acid sequences of the CD22 isoforms (from top to bottom SEQ ID NO// to SEQ ID NO//). Figure 9A shows a site map showing possible sites of modification for the generation of aldehyde-tagged Ig polypeptides. The top sequence is the amino acid sequence (SEQ ID NO://) of the conserved region of IgG1 light chain polypeptides, showing possible modification sites in the Ig light chain, and the bottom sequence is the conserved region of Ig heavy chain polypeptides. Amino acid sequence (SEQ ID NO://; GenBank Accession No. AAG00909) showing possible modification sites in the Ig heavy chain. Heavy and light chain numbering is based on full-length heavy and light chains. FIG. 9B depicts the immunoglobulin heavy chain constant regions of IgG1 (SEQ ID NO//), IgG2 (SEQ ID NO//), IgG3 (SEQ ID NO//), IgG4 (SEQ ID NO//) and IgA (SEQ ID NO//). Alignments are shown and modification sites at which aldehyde tags can be provided in the immunoglobulin heavy chain are shown. Heavy and light chain numbering is based on complete heavy and light chains. FIG. 9C shows an alignment of immunoglobulin light chain constant regions (SEQ ID NO://) showing modification sites at which aldehyde tags may be provided in the immunoglobulin light chain. FIG. 10 shows an illustration of an ELISA format for detection of various analytes according to embodiments of the present disclosure. The anti-CD22 ADC of the present disclosure was highly monomeric, had an average DAR of 1.8, and contained single light and heavy chain species. by size exclusion chromatography (Figure 11 panel A) to assess the monomer ratio (99.2%) and to assess the drug-to-antibody ratio (DAR), which was 1.8. Anti-CD22 ADCs were evaluated by hydrophobic interaction (HIC; Figure 11 panel B) and reverse phase (PLRP) chromatography (Figure 11 panel C). The anti-CD22 ADCs of the present disclosure bound human CD22 protein equivalently to wild-type anti-CD22 antibodies. A competitive ELISA was used to compare the binding of anti-CD22 ADCs to wild-type (WT) anti-CD22 antibodies. Data are mean ± S.E.M. D. (n=4). Anti-CD22 ADCs of the disclosure mediated CD22 internalization similarly to wild-type anti-CD22 antibodies. NHL cell lines, Ramos, Granta-519 and WSU-DLCL2, were used to compare internalization of cell surface CD22 mediated by binding to either WT anti-CD22 or CAT-02-106. Anti-CD22 ADCs of the disclosure were equally effective in vitro against parental and MDR1-expressing NHL tumor cells. Ramos and WSU-DLCL2 parental (WT) cells (FIG. 14 panels A and C), and mutants of those lines engineered to express MDR1 (MDR1+; FIG. 14 panels B and D) were treated with anti-CD22. It was used as a target for in vitro cytotoxicity testing of ADC activity. αCD22 ADC generated using the CAT-02 antibody but conjugated to maytansine using a valine-citrulline cleavable linker and free maytansine were used as controls. In additional control experiments, the MDR1 inhibitor cyclosporine was added to WT or MDR1+ WSU-DLCL2 cells (FIG. 14 panels E and F). Data are mean ± S.E.M. D. (n=2). Anti-CD22 ADCs of the disclosure did not mediate off-target cytotoxicity. Gastric tumor cell line NCI-N87 was incubated in vitro for 5 days in the presence of increasing concentrations of anti-CD22 ADC. Cell viability was then assessed using an MTS-based method. Data are mean ± S.E.M. D. (n=2). An anti-CD22 ADC-related ADC, anti-HER2 conjugated to the HIPS-4AP-maytansine linker payload, did not induce bystander killing. In vitro cytotoxicity studies were performed using HER2+ NCI-N87 cells, HER2- Ramos cells or co-cultures of both cells as targets. Anti-HER2 conjugated to MMAE via a cleavable valine-citrulline (vc) linker (2 nM payload) and free maytansine (2 nM) were used as positive controls for bystander killing. Anti-HER2 ADC was administered at 2 nM payload. Data are mean ± S.E.M. D. (n=2). Anti-CD22 ADCs of the disclosure were effective in vivo against NHL-derived WSU-DLCL2 and Ramos xenograft models. anti-CD22 ADC as a single 10 mg/kg dose (Figure 17 panel A) or as multiple 10 mg/kg doses administered every 4 days for a total of 4 doses (q4d x 4), or vehicle alone. , treated female CB17 ICR SCID mice (8/group) containing WSU-DLCL2 xenografts. Treatment was initiated when tumors reached an average size of 118 or 262 mm ³ for the single or multiple dose studies, respectively. (FIG. 17 panel C): Female CB17 ICR SCID mice (12/group) containing Ramos xenografts were treated with vehicle alone or 5 or 10 mg/kg CAT-02-106 q4d×4. Dosing was initiated when tumors reached an average size of 246 mm ³ . Data are mean ± S.E.M. E. M. is shown as Ramos and WSU-DLCL2 cells expressed different levels of cell surface CD22. Ramos and WSU-DLCL2 cells were incubated with fluorescein-labeled anti-CD22 antibody and then analyzed by flow cytometry. The mean fluorescence intensity of the FL1 channel (detecting fluorescein) for each cell type is shown in the graph. Mouse body weight was not affected by treatment with the disclosed anti-CD22 ADCs. Mean body weights of mice in xenograft efficacy studies are shown. (Figure 19 panel A) single dose WSU-DLCL2 study; (Figure 19 panel B) multiple dose WSU-DLCL2 study; (Figure 19 panel C) Ramos study. Error bars are S.E. D. indicates Anti-CD22 ADCs of the present disclosure can be administered in rats up to 60 mg/kg with minimal effect. Sprague-Dawley rats (5/group) were administered 6, 20, 40 or 60 mg/kg doses of CAT-02-106 followed by a 12 day observation period. (Figure 20 panel A) Body weight was monitored at the indicated time points. (Figure 20 panel B) Alanine aminotransferase (ALT) and (Figure 20 panel C) platelet counts were assessed on days 5 and 12 after dosing. Data are mean ± S.E.M. D. is shown as Anti-CD22 ADCs of the disclosure specifically bound to cynomolgus B cells. Cynomolgus monkey peripheral blood lymphocytes were gated according to their forward and side scatter profiles (upper left). Cells were incubated in the presence of fluorescein-isothiocyanate (FITC)-conjugated streptavidin (SA) alone (top right) or biotinylated anti-CD22 ADC followed by FITC SA. Co-incubation with antibodies recognizing T cells (CD3, bottom left) or B cells (CD20, bottom right) demonstrated the specificity of CAT-02-106 binding to B cell populations. Anti-CD22 ADCs of the disclosure demonstrated B-cell specific reactivity in human and cynomolgus tissue. Anti-CD22 ADC bound to the B cell-rich region of the spleen (top). Heart tissue was negative for staining (middle). Except for scattered leukocytes, the lung section was negative (bottom). No side effects are observed at repeated doses of 60 mg/kg of anti-CD22 ADCs of the disclosure in cynomolgus monkeys. Cynomolgus monkeys (2/sex/group) were administered 10, 30 or 60 mg/kg of anti-CD22 ADC once every 3 weeks for a total of 2 doses followed by a 21 day observation period. (Figure 23 panel A) aspartate transaminase (AST), (Figure 23 panel B) alanine aminotransferase (ALT), (Figure 23 panel C) platelets, and (Figure 23 panel D) monocytes at the indicated time points. monitored by Data are mean ± S.E.M. D. is shown as (Panel A and Panel B)—Treatment with anti-CD22 ADCs of the present disclosure decreased peripheral B cell populations in cynomolgus monkeys. The ratios of B cells (CD20+), T cells (CD3+) and NK cells (CD20-/CD3-) observed in the animals before dosing and on days 7, 14, 28 and 35 were For detection, peripheral blood mononuclear cells from cynomolgus monkeys enrolled in the toxicity study were monitored by flow cytometry. Data are mean ± S.E.M. D. is shown as The anti-CD22 ADCs of the present disclosure exhibited very high in vivo stability as demonstrated by rat pharmacokinetic studies. Sprague-Dawley rats (3/group) were dosed with a single i.v. v. A bolus dose of anti-CD22 ADC was administered. Plasma samples were collected at the indicated time points and analyzed for total antibody, total conjugate and total ADC concentrations (as shown in Figure 10). FIG. 26 shows Table 3, which shows mean (±SD) pharmacokinetic parameters and toxicokinetic (TK) total ADC values in animals dosed with anti-CD22 ADCs in various embodiments of the present disclosure. A summary of parameters.

EXAMPLES The following examples are included to provide those skilled in the art with a complete disclosure and illustration of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, and ,
Nor is it intended that the experiments described below were the only or all experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.);
Some experimental error and deviation should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. "Average" means arithmetic mean. For example, standard abbreviations such as the following may be used: bp, base pairs; kb, kilobases; pl, picoliters; s or s
ec, seconds; min, minutes; h or hr, hours; aa, amino acids; kb, kilobases;
, base pairs; nt, nucleotides; i. m. , intramuscularly; i. p. , intraperitoneal; s. c. , subcutaneous;

GENERAL SYNTHETIC METHODS Numerous general references are available (e.g., Smith and March
, March's Advanced Organic Chemistry: Reac
ions, Mechanisms, and Structure, Fifth Edi
tion, Wiley-Interscience, 2001; or Vogel, A.
Textbook of Practical Organic Chemistry,
Including Qualitative Organic Analysis, F.
outer Edition, New York: Longman, 1978).

The compounds described herein can be purified by any purification method known in the art, including chromatography such as HPLC, preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases and ionic resins. In some embodiments, the disclosed compounds are purified by silica gel and/or alumina chromatography. For example, Introduction to Modern Liquid
Chromatography, 2nd Edition, L.L. R. Snyder andJ. J. Kirkland, ed., John Wiley and Sons, 1979; and Thin Layer Chromatography, E.M. Ed. Stahl, Sp.
See Ringer-Verlag, New York, 1969.

During any of the methods of preparing the compounds it may be necessary and/or desirable to protect sensitive or reactive groups on any of the subject molecules. This can be done using conventional protecting groups as described in standard research documents such as: J. Am.
F. W. McOmie, “Protective Groups in Organic
Chemistry”, Plenum Press, London and New
York 1973,T. W. Greene and P.S. G. M. Wuts, “Prote
Active Groups in Organic Synthesis", Third
edition, Wiley, New York 1999, "The Peptid
es"; Volume 3 (E. Gross and J. Meienhofer, eds.), Ac.
academic Press, London and New York 1981, "M
Ethoden der organischen Chemie”, Houben-W.
eyl, 4th edition, Vol. 15/l, Georg Thieme Ve
rlag, Stuttgart 1974, H.R. -D. Jakubke and H. Jesus
Cheit, "Aminosauren, Peptide, Protein", Ver.
Lag Chemie, Weinheim, Deerfield Beach and Ba
sel 1982, and/or Jochen Lehmann, "Chemie d
er Kohlenhydrate: Monosaccharide and Deri
vate", Georg Thieme Verlag, Stuttgart 1974
. Protecting groups may be removed in convenient subsequent steps using methods known in the art.

The compounds may be synthesized by a variety of synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. Various examples of synthetic routes that can be used to synthesize the compounds disclosed herein are described in the schemes below.

Example 1
A linker containing a 4-amino-piperidine (4AP) group was synthesized according to Scheme 1 shown below.

Synthesis of (9H-fluoren-9-yl)methyl 4-oxopiperidine-1-carboxylate (200) Piperidin-4-one hydrochloride monohydrate (1) was added to a 100 mL round bottom flask containing a magnetic stir bar.
. 53 g, 10 mmol), Fmoc chloride (2.58 g, 10 mmol), sodium carbonate (3.18 g, 30 mmol), dioxane (20 mL) and water (2 mL) were added. The reaction mixture was stirred at room temperature for 1 hour. The mixture was diluted with EtOAc (100 mL) and extracted with water (1 x 100 mL). The organic layer was dried over _Na2SO4 , _filtered and concentrated under reduced pressure. The resulting material was dried in vacuo to give compound 200 as a white solid (3
. 05 g, 95% yield).

¹ H NMR (CDCl ₃ ) δ 7.78 (d, 2H, J=7.6), 7.59 (d,
2H, J = 7.2), 7.43 (t, 2H, J = 7.2), 7.37 (t, 2H, J = 7
. 2), 4.60 (d, 2H, J=6.0), 4.28 (t, 2H, J=6.0), 3.
72 (br, 2H), 3.63 (br, 2H), 2.39 (br, 2H), 2.28 (b
r, 2H).

MS ₍ ESI) m/z: _calc'd for [M+H] ⁺ _C20H20NO3 : 322.
4; Found: 322.2.

Synthesis of (9H-fluoren-9-yl)methyl 4-((2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)amino)piperidine-1-carboxylate (201) Piperidinone 200 (642 mg,
2.0 mmol), H ₂ N-PEG ₂ -CO ₂ t-Bu (560 mg, 2.4 mmol)
), 4 Angstrom molecular sieves (active powder, 500 mg) and 1,2-dichloroethane (5 mL) were added. The mixture was stirred at room temperature for 1 hour. Sodium triacetoxyborohydride (845 mg, 4.0 mmol) was added to the reaction mixture. The mixture was stirred at room temperature for 5 days. The resulting mixture was diluted with EtOAc. The organic layer is saturated NaHCO
₃ (1 x 50 mL) and brine ( ₁ x 50 mL), dried over _Na2SO4 , filtered, and concentrated under reduced pressure to give compound 201 as an oil, which was further purified. I proceeded to the next step.

13-(1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidin-4-yl)-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-1
Synthesis of 3-azaheptadecan-17-acid (202) N-Fmoc-piperidine-4-amino-PEG ₂ -CO ₂ t-Bu (201), succinic anhydride from the previous step was added to a dry scintillation vial containing a magnetic stir bar. (270mg
, 2.7 mmol) and dichloromethane (5 mL) were added. The mixture was stirred at room temperature for 18 hours. The reaction mixture was partitioned between EtOAc and saturated _NaHCO3 . The aqueous layer was diluted with EtO
Extracted with Ac (3x). The aqueous layer was acidified with HCl (1M) until pH˜3. The aqueous layer was extracted with DCM (3x). The combined organic layers _were dried over _Na2SO4 , filtered and concentrated under reduced pressure. The reaction mixture was purified by C18 flash chromatography (eluting with 10-100% MeCN/water containing 0.1% acetic acid). Product-containing fractions were concentrated under reduced pressure and then azeotroped with toluene (3 x 50 mL) to remove residual acetic acid, yielding 534 mg (
42%, 2 steps) of compound 202 was obtained as a white solid.

¹ H NMR (DMSO-d ₆ ) δ 11.96 (br, 1H), 7.89 (d, 2H
, J=7.2), 7.63 (d, 2H, J=7.2), 7.42 (t, 2H, J=7.2
), 7.34 (t, 2H, J = 7.2), 4.25-4.55 (m, 3H), 3.70-
4.35 (m, 3H), 3.59 (t, 2H, J = 6.0), 3.39 (m, 5H), 3
. 35 (m, 3H), 3.21 (br, 1H), 2.79 (br, 2H), 2.57 (m
, 2H), 2.42 (q, 4H, J = 6.0), 1.49 (br, 3H), 1.37 (s
, 9H).

MS (ESI) m/z: _calc'd for [M ₊ H] ⁺ _{C35H47N2O 9} : 639.
. 3; Found: 639.2.

(2S)-1-((1 ⁴ S, 1 ⁶ S, 3 ³ S, 2R, 4S, 10E, 12E, 14R)
-8 ⁶ -chloro-1 ⁴ -hydroxy-8 ⁵ ,14-dimethoxy-3 ³ ,2,7,10-tetramethyl-1 ² ,6-dioxo-7-aza-1(6,4)-oxazinana- 3 (2,3
)-oxirana-8(1,3)-benzeneacyclotetradecaphan-10,12-dien-4-yl)oxy)-2,3-dimethyl-1,4,7-trioxo-8-(piperidine- 4-yl)-11,14-dioxa-3,8-diazaheptadecan-17-acid (20
Synthesis of 3) Ester 202 (227 mg, 0.356 mmol), diisopropylethylamine (174 μL, 1.065 mmol), N-deacetylmaytansine 1 in 2 mL DMF
PyAOP (185 mg, 0.355 mg) was added to a solution of 24 (231 mg, 0.355 mmol).
mmol) was added. The solution was stirred for 30 minutes. Piperidine (0.5 mL) was added to the reaction mixture and stirred for an additional 20 minutes. The crude reaction mixture was purified by C18 reverse-phase chromatography using a 0-100% acetonitrile:water gradient to yield 203.2 mg (55%,
2 step) to give compound 203.

17-(tert-butyl) 1-((1 ⁴ S, 1 ⁶ S, 3 ³ S, 2R, 4S, 10E
, 12E, 14R)-8 ⁶ -chloro-1 ⁴ -hydroxy-8 ⁵ ,14-dimethoxy-3 ³
,2,7,10-tetramethyl-1 ² ,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzeneacyclotetra Decafane-10,12-dien-4-yl)(2S)-8-(1-(3-(2-((2-(((9
H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)
methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-2,
Synthesis of 3-dimethyl-4,7-dioxo-11,14-dioxa-3,8-diazaheptadecanedioate (204) Piperidine 203 (203.2 mg, 0.194 mmol) in 1 mL of DMF, ester 12 ( 126.5 mg, 0.194 mmol), 2,4,6-trimethylpyridine (
77 μL, 0.582 mmol), HOAT (26.4 mg, 0.194 mmol) was stirred for 30 minutes. The crude reaction was purified by C18 reverse-phase chromatography using a gradient of 0-100% acetonitrile:water containing 0.1% formic acid to give 280.5 mL
g (97% yield) of compound 204 was obtained.

MS (ESI) m/z: _calc'd for [M+H] ^< +> _{C81H106ClN8O18} : _1513.7 ; found: _1514.0 .

(2S)-8-(1-(3-(2-((2-((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indole- 1-
yl)propanoyl)piperidin-4-yl)-1-(((1 ⁴ S, 1 ⁶ S, 3 ³ S, 2
R,4S,10E,12E,14R)-8 ⁶ -chloro-1 ⁴ -hydroxy-8 ⁵ ,14-
Dimethoxy-3 ³ ,2,7,10-tetramethyl-1 ² ,6-dioxo-7-aza-1 (
6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzeneacyclotetradecaphan-10,12-dien-4-yl)oxy)-2,3-dimethyl-1 ，
4,7-trioxo-11,14-dioxa-3,8-diazaheptadecan-17-acid (
Synthesis of 205) To a solution of compound 204 (108 mg, 0.0714 mmol) in 500 μL of anhydrous DCM was added 357 μL of a 1 M solution of SnCl ₄ in DCM. The heterogeneous mixture was stirred for 1 hour and then washed with a gradient of 0-100% acetonitrile:water containing 0.1% formic acid.
18 Purification by reverse phase chromatography gave 78.4 mg (75% yield) of compound 20.
Got 5.

MS ₍ ESI) m/z ^: _calcd for [ _MH ] _{-C77H96ClN8O18} :
1455.7; Found: 1455.9.

Example 2
A linker containing a 4-amino-piperidine (4AP) group was synthesized according to Scheme 2 shown below.

Synthesis of tert-butyl 4-oxopiperidine-1-carboxylate (210) Piperidin-4-one hydrochloride monohydrate (
1.53 g, 10 mmol), di-tert-butyl dicarbonate (2.39 g, 11
mmol), sodium carbonate (1.22 g, 11.5 mmol), dioxane (10 mL
) and water (1 mL) were added. The reaction mixture was stirred at room temperature for 1 hour. Add the mixture to water (10
0 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers _were washed with brine, dried over _Na2SO4 , filtered and concentrated under reduced pressure. The resulting material was dried in vacuo to yield 1.74 g (87%) of compound 210 as a white solid.

¹ H NMR (CDCl3) δ 3.73 (t, 4H, J = 6.0), 2.46 (t,
4H, J=6.0), 1.51 (s, 9H).

MS (ESI) m/z: _{calc'd for} [M+H] ⁺ _C10H18NO3 : 200.
3; Found value: 200.2.

tert-butyl 4-((2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)amino)piperidine-1-carboxylate (211)
In a dry scintillation vial containing a magnetic stir bar was added tert-butyl 4-oxopiperidine-1-carboxylate (399 mg, 2 mmol), H ₂ N-PEG ₂ -COO.
t-Bu (550 mg, 2.4 mmol), 4 Angstrom molecular sieves (
Activated powder, 200 mg) and 1,2-dichloroethane (5 mL) were added. The mixture was stirred at room temperature for 1 hour. Sodium triacetoxyborohydride (845 m) was added to the reaction mixture.
g, 4 mmol) was added. The mixture was stirred at room temperature for 3 days. The resulting mixture was treated with EtOAc
and saturated aqueous _NaHCO3 . The organic layer is washed with brine and Na ₂ SO ₄
, filtered and concentrated under reduced pressure to give 850 mg of compound 211 as a viscous oil.

MS (ESI) m/z: _calc'd for [M ₊ H] ⁺ _C21H41N2O6 : ₄₁₇ .
. 3; Found: 417.2.

13-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadecane-1
7-acid (212)
Tert-Butyl 4-((2-(
2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)amino)piperidine-1-carboxylate 211 (220 mg, 0.5 mmol), succinic anhydride (55 mg, 0.55 mmol), 4- (dimethylamino)pyridine (5 mg, 0.
04 mmol) and dichloromethane (3 mL) were added. The mixture was stirred at room temperature for 24 hours. The reaction mixture was partially purified by flash chromatography (eluting with 50-100% EtOAc/hexanes) to give 117 mg of compound 212 as a clear oil,
This was carried on without further characterization.

MS (ESI) m/z: _calc'd for [M ₊ H] ⁺ _{C25H45N2O 9} : 517.
. 6; Found: 517.5.

17-(tert-butyl) 1-((1 ⁴ S, 1 ⁶ S, 3 ³ S, 2R, 4S, 10E
, 12E, 14R)-8 ⁶ -chloro-1 ⁴ -hydroxy-8 ⁵ ,14-dimethoxy-3 ³
,2,7,10-tetramethyl-1 ² ,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzeneacyclotetra Decafane-10,12-dien-4-yl)(2S)-8-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2,3-dimethyl-4,7-dioxo-11,14 -Dioxa-3,8-diazaheptadecanedioate (213) 13-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2,2-dimethyl in a dry scintillation vial containing a magnetic stir bar. -4,14-dioxo-3,7
, 10-trioxa-13-azaheptadecan-17-acid 212 (55 mg, 0.1 mm
ol), N-deacylmaytansine 124 (65 mg, 0.1 mmol), HATU (4
3 mg, 0.11 mmol), DMF (1 mL) and dichloromethane (0.5 mL) were added. The mixture was stirred at room temperature for 8 hours. The reaction mixture was purified directly by C18 flash chromatography (eluting with 5-100% MeCN/water) to give 18 mg (16%) of compound 213 as a white film.

MS _{( ESI} ) m/z: _calcd for [M+H] ^< +> _{C57H87ClN5O17} :
1148.6; Found: 1148.7.

(2S)-1-(((1 ⁴ S, 1 ⁶ S, 3 ³ S, 2R, 4S, 10E, 12E, 14R
)-8 ⁶ -chloro-1 ⁴ -hydroxy-8 ⁵ ,14-dimethoxy-3 ³ ,2,7,10-
Tetramethyl-1 2,6-dioxo-7-aza-1(6,4)-oxazinana-3( ² ,
3)-oxirana-8(1,3)-benzeneacyclotetradecaphan-10,12-dien-4-yl)-2,3-dimethyl-1,4,7-trioxo-8-(piperidine-4
-yl)-11,14-dioxa-3,8-diazaheptadecan-17-acid (214) Maytansinoid 213 (31 mg) was added to a dry scintillation vial containing a magnetic stir bar.
, 0.027 mmol) and dichloromethane (1 mL) were added. The solution was cooled to 0° C. and tin(IV) tetrachloride (1.0 M in dichloromethane, 0.3 mL, 0.3 mmol)
was added. The reaction mixture was stirred at 0° C. for 1 hour. The reaction mixture was purified directly by C18 flash chromatography (eluting with 5-100% MeCN/water) to give 16 mg (60%)
was obtained as a white solid (16 mg, 60% yield).

MS _{( ESI} ) m/z: _calcd for [M+H] ^< +> _{C48H71ClN5O15} :
992.5; Found: 992.6.

(2S)-8-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indole -1
-yl)propanoyl)piperidin-4-yl)-1-(((1 ⁴ S, 1 ⁶ S, 3 ³ S,
2R,4S,10E,12E,14R)-8 ⁶ -chloro-1 ⁴ -hydroxy-8 ⁵ ,14
-dimethoxy-3 ³ ,2,7,10-tetramethyl-1 ² ,6-dioxo-7-aza-1
(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzeneacyclotetradecaphan-10,12-dien-4-yl)-2,3-dimethyl-1, 4,7
-trioxo-11,14-dioxa-3,8-diazaheptadecan-17-acid (215
) Maytansinoid 214 (16 mg) was added to a dry scintillation vial containing a magnetic stir bar.
, 0.016 mmol), (9H-fluoren-9-yl)methyl 1,2-dimethyl-
2-((1-(3-oxo-3-(perfluorophenoxy)propyl)-1H-indol-2-yl)methyl)hydrazine-1-carboxylate (5) (13 mg, 0.0
2 mmol), DIPEA (8 μL, 0.05 mmol) and DMF (1 mL) were added. The solution was stirred at room temperature for 18 hours. The reaction mixture was purified directly by C18 flash chromatography (eluting with 5-100% MeCN/water) to give 18 mg (77%) of compound 215 as a white solid.

MS _{( ESI} ) m/z: _calcd for [M+H] ⁺ _{C77H98ClN8O18} :
1457.7; Found: 1457.9.

(2S)-1-(((1 ⁴ S, 1 ⁶ S, 3 ³ S, 2R, 4S, 10E, 12E, 14R
)-8 ⁶ -chloro-1 ⁴ -hydroxy-8 ⁵ ,14-dimethoxy-3 ³ ,2,7,10-
Tetramethyl-1 2,6-dioxo-7-aza-1(6,4)-oxazinana-3( ² ,
3)-oxirana-8(1,3)-benzeneacyclotetradecaphan-10,12-dien-4-yl)oxy)-8-(1-(3-(2-((1,2-dimethyl) hydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-2
Synthesis of ,3-dimethyl-1,4,7-trioxo-11,14-dioxa-3,8-diazaheptadecan-17-acid (216) 18mg
, 0.012 mmol), piperidine (20 μL, 0.02 mmol) and DMF (1
mL) was added. The solution was stirred at room temperature for 20 minutes. The reaction mixture was purified directly by C18 flash chromatography (eluting with 1-60% MeCN/water) to give 15 mg (98
%) of compound 216 (herein HIPS-4AP-maytansine or HIP
S-4-amino-piperidine-maytansine) was obtained as a white solid.

MS _{( ESI} ) m/z: _calcd for [M+H] ^< +> _{C62H88ClN8O16} :
1235.6; Found: 1236.0.

Example 3
Experimental Procedures General Experiments were performed to produce site-specific conjugated antibody-drug conjugates (ADCs). Production of site-specific ADCs requires the unnatural amino acid formylglycine (FG
ly) into the protein sequence. To introduce FGly (Fig. 1), the short consensus sequence CXPXR (where X is serine, threonine, alanine or glycine) was cloned into antibody heavy chains using standard molecular biology cloning techniques. Or inserted at the desired position within the conserved region of the light chain. This "tagged" construct was obtained recombinantly in cells co-expressing formylglycine-generating enzyme (FGE). FGE
co-translationally converted a cysteine in the tag to an FGly residue to provide an aldehyde functional group (also referred to herein as an aldehyde tag). The aldehyde functional group served as a chemical handle for bioorthogonal conjugation. A payload (such as a drug, such as a cytotoxin (such as maytansine)) is attached to FGly using a hydrazino-iso-pictet-Spengler (HIPS) ligation to create a stable covalent bond between the cytotoxin payload and the antibody, C- A C bond was formed. This C-
The C-bond was expected to be stable to physiologically relevant conditions encountered by ADCs during circulation and FcRn recycling, such as proteases, low pH and reducing reagents. Antibodies containing aldehyde tags can be produced in a variety of locations. Experiments were performed to test the effect of inserting an aldehyde tag at the heavy chain C-terminus (CT). Biophysical and functional characterization was performed on the resulting ADCs produced by conjugation to the maytansine payload via the HIPS linker.

Cloning, Expression and Purification of Tagged Antibodies An aldehyde tag sequence was inserted at the heavy chain C-terminus (CT) using standard molecular biology techniques. For small-scale production, CHO-S cells were transfected with a human FGE expression construct and pools of FGE-overexpressing cells were used for transient production of antibody. For larger scale production, GPEx technology (Catalent, Inc., Somerset, NJ) was used to obtain a clonal cell line overexpressing human FGE (GPEx). Then, the FG
E clones were used to obtain bulk stable pools of antibody-expressing cells. Protein A chromatography (MabSelect, GE Healthcare Life Science
Antibodies were purified from the conditioned medium using ces, Pittsburgh, Pa.). Purified antibodies were flash frozen and stored at -80°C until further use.

Bioconjugation, purification and HPLC analysis 50 mM sodium citrate containing 0.85% DMA, 50 mM NaCl (
HIP C-terminal aldehyde-tagged αCD22 antibody (15 mg/mL) in pH 7.4)
Conjugated to S-4AP-maytansine (8 molar equivalents of drug:antibody) at 37° C. for 72 hours. Mobile phase A: 1.0 M ammonium sulfate, 25 mM sodium phosphate (pH
7.0) and mobile phase B: 25% isopropanol, 18.75 mM sodium phosphate, pH 7.0, preparative-scale hydrophobic interaction chromatography (HIC
GE Healthcare 17-5195-01) was used to remove unconjugated antibody. An isocratic gradient of 33% B was used to elute unconjugated material, followed by 41-95 to elute mono- and di-conjugated species.
A linear gradient of % B was used. To determine the DAR of the final product, mobile phase A: 1.5M
ammonium sulfate, 25 mM sodium phosphate (pH 7.0) and mobile phase B: 25
ADC was examined by analytical HIC (Tosoh #14947, Grove City, OH) using % isopropanol, 18.75 mM sodium phosphate, pH 7.0. To measure aggregation, 300 mM NaCl, 25 mM sodium phosphate (pH
6.8) Analytical size exclusion chromatography (SEC; Tosoh
#08541) was used to analyze the samples.

Results αCD22 antibody modified to contain an aldehyde tag at the heavy chain C-terminus (CT) was
It was conjugated to the maytansine payload attached to the HIPS-4AP linker described above. Upon completion of the conjugation reaction, unconjugated antibody was removed by preparative HIC and residual free drug was removed by tangential flow filtration during buffer exchange.
These reactions are highly yielding, with conjugation efficiencies greater than 84% and an overall yield of 70%.
%. The resulting ADCs were predominantly monomeric with a drug-to-antibody ratio (DAR) of 1.6-1.9. Figures 2-5 show DAR from representative crude reactions and purified ADC as determined by HIC and reverse-phase PLRP chromatography, and demonstrate monomer integrity as determined by SEC.

Figure 2 shows the C-terminal (CT
) shows a hydrophobic interaction column (HIC) trace of an aldehyde-tagged anti-CD22 antibody conjugated in ). Figure 2 shows the crude D determined by HIC.
It shows that the AR was 1.68.

Figure 3 shows the C-terminal (CT
) shows HIC traces of aldehyde-tagged anti-CD22 antibodies conjugated in . Figure 3 shows that the final DAR determined by HIC was 1.77.

Figure 4 shows the C-terminal (CT
) shows reverse phase chromatography (PLRP) traces of aldehyde-tagged anti-CD22 antibodies conjugated in . Figure 4 shows that the final DAR determined by RLRP was 1.81.

Figure 5 shows the C-terminal (CT
) shows a graph of analytical size exclusion chromatography (SEC) analysis of conjugated aldehyde-tagged anti-CD22 antibodies in FIG. As shown in Figure 5, analytical SEC showed 98.2% monomer for the final product.

In Vitro Cytotoxicity The CD22-positive B-cell lymphoma cell lines Ramos and WSU-DLCL2 were obtained from the ATCC and DSMZ cell banks, respectively. The cells were added with 10% fetal bovine serum (Invitrogen, Grand Island, NY) and Glutamax (
RPMI-1640 medium (Cellgro, Mann.) supplemented with Invitrogen)
assas, VA). Cells were passaged to ensure exponential growth 24 hours prior to plating. On the day of plating, 5000 cells/well were
96-well plates were seeded in 90 μL of normal growth medium supplemented with U penicillin and 10 μg/mL streptomycin (Cellgro). Cells were treated with 10 μL of diluted analyte at various concentrations and plates were incubated at 37° C. in an atmosphere of 5% CO ₂ . After 5 days, 100 μL/well of Cell Titer-Glo reagent (P
romega, Madison, Wis.) was added and obtained from Molecular Devices.
Luminescence was measured using a SpectraMax M5 plate reader. GraphP
ad Prism software was used for data analysis.

Results αCD22 CT HIPS-4AP-maytansine showed very potent in vitro activity against WSU-DLCL2 and Ramos cells compared to free maytansine (FIG. 6). The _IC50 concentrations were 0.018 and 0.086 nM for ADC and free drug, respectively, against WSU-DLCL2 cells and 0.007 and 0 for ADC and free drug, respectively, against Ramos cells. 0.040 nM.

FIG. 6A shows the C-terminal (C
In T), a graph showing the in vitro potency [Log antibody drug conjugate (ADC) concentration (nM) versus viability (%)] against WSU-DLCL2 cells for conjugated anti-CD22 ADCs is shown. FIG. 6B Anti-CD22 conjugated at the C-terminus (CT) to a maytansine payload linked to a HIPS-4AP linker.
FIG. 3 shows a graph showing in vitro potency of ADC against Ramos cells [Log antibody drug conjugate (ADC) concentration (nM) vs. viability (%)].

Xenograft Studies Female ICR SCID mice (8/group) were inoculated subcutaneously with 5×10 ⁶ WSU-DLCL2 cells. Treatment began when tumors reached an average of 262 mm ³ at which time vehicle alone or CT-tagged αCD22HIPS-4AP-maytansine (10 mg/kg) was administered.
was administered intravenously to animals. Dosing was performed every 4 days for a total of 4 doses (q4d x 4 times). Animals were monitored twice weekly for body weight and tumor size. Animals were euthanized when tumors reached 2000 mm ³ .

Results The median time to endpoint for animals in the vehicle control group was 16 days.
Therefore, the tumor growth inhibition rate (TGI%) was calculated on that day. TGI% was defined by the following formula.

TGI (%) = (TV _{control group} - TV _{treatment group} ) / TV _control x 100
where TV is the tumor volume.

Animals receiving αCD22 HIPS-4AP-maytansine had a T of 90% on day 16
showed GI and 5 out of 8 tumors showed complete regression (Fig. 7). Three of these complete regressions were sustainable until the end of the study (Day 58). FIG. 7. In vivo efficacy [mean tumor volume over days (mm ³ )] against WSU-DLCL2 xenograft model for anti-CD22 ADC conjugated at the C-terminus (CT) to a maytansine payload attached to a HIPS-4AP linker. shows a graph showing Vertical arrows in Figure 7 indicate dosing, which was performed every 4 days for a total of 4 doses (q4d x 4).

Example 4
INTRODUCTION Bloodborne tumors account for approximately 10% of all newly diagnosed cancer cases in the United States. Of these, the name non-Hodgkin lymphoma (NHL) is one of the most common diagnoses worldwide.
The diverse groups ranked together within 0 cancers are shown. Although long-term survival trends are improving, there is still a lack of satisfaction with treatments to help patients with relapsed or refractory disease, partly due to drug excretion through upregulation of xenobiotic pumps such as MDR1. There are still significant unmet clinical needs. MDR
A site-specific conjugated antibody-drug conjugate targeted against CD22 containing a non-cleavable maytansine payload that is resistant to 1-mediated efflux was prepared. The construct is efficacious against CD22+ NHL xenografts, with no side effects observed.
Repeat doses can be given in cynomolgus monkeys at 0 mg/kg. Taken together, the data indicated that this drug has potential to be used effectively in patients with CD22+ tumors who have developed MDR1-associated resistance to previous therapies. CD22 is the NHL
and clinically validated targets for the treatment of ALL. Anti-CD22 antibody-drug conjugates (ADCs) according to the present disclosure can be used to treat relapsed/refractory NHL and ALL patients.

Materials and Methods An anti-CD22 antibody was site-specifically conjugated to a non-cleavable maytansine payload using aldehyde tag technology. The ADC was characterized both biophysically and functionally in vitro. In vivo efficacy was then determined using two xenograft models in mice and toxicity studies were performed in both rats and cynomolgus monkeys. Pharmacodynamic studies were conducted in monkeys and pharmacokinetic and toxicokinetic studies compared to total ADC exposure in the efficacy and toxicity studies.

Results The ADC was highly potent in vivo, even against cell lines engineered to overexpress the efflux pump MDR1. This construct is 1
Effective at 0 mg/kg x 4 doses, in cynomolgus monkey toxicity studies, the ADC
was administered twice at 60 mg/kg and no side effects were observed. Total A at these doses
Exposure to DC (as assessed by AUC _0-inf ) indicated that the exposure required to achieve efficacy was below the permissible limit. Finally, studies of pharmacodynamic responses in treated monkeys showed that the B-cell compartment was selectively depleted (depleted), indicating that ADCs could be administered without significant off-target toxicity. , indicating exclusion of target cells.

These results indicated that the ADC could be used effectively in patients with CD22+ tumors who had developed MDR1-associated resistance to previous therapies.

Example 5
INTRODUCTION Leukemia, lymphoma and myeloma are very common in the population, accounting for approximately 10% of all newly diagnosed cancer cases in the United States in 2015. Among these cancers, B-cell derived malignancies constitute a large and diverse group including non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). Similarly, as a category, NHL includes about 60 lymphoma subsets, of which about 85% are of B-cell origin, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL ) and mantle cell lymphoma (MCL). Taken together, NHL disease is the most common cancer type observed and was the 10th most common cancer diagnosed worldwide in 2012.
And it ranks as the seventh most common cancer in the United States. Although long-term trends show improvement in 5-year survival rates for most haematological cancer diagnoses, there is still a significant unmet clinical need, diagnosed between 2004 and 2010. 16% of CLL patients
, 30% of ALL patients and 30% of NHL patients do not meet the 5-year survival endpoint.

CD22 is one of the B-lineage restricted cell surface glycoproteins, which is expressed on the majority of B-cell hematological malignancies, but not on hematopoietic stem cells, memory B-cells or other normal non-hematopoietic tissues. Not expressed. Its expression pattern and rapid internalization kinetics make it a target for antibody-drug conjugate (ADC) therapy, which is the NH
It has been validated as such in clinical trials for L and ALL.

In the experiments described herein, the aldehyde tag and hydrazino-iso-
A site-specific conjugation technique based on Pictet-Spengler (HIPS) chemistry was used to place the maytansine payload attached to the antibody heavy chain C-terminus via a non-cleavable linker. A genetically encoded aldehyde tag incorporated the 6 amino acid sequence LCTPSR. In co-translation, overexpressed formylglycine-generating enzyme (FGE) converted cysteines within the consensus sequence to formylglycine residues with aldehyde functional groups. H this
The ADC was obtained by reaction with the IPS linker payload. This approach provided control over both payload placement and DAR, yielding highly homogeneous ADC preparations. Site-specifically conjugated ADCs provided improved pharmacokinetics (PK) and efficacy compared to stochastic conjugates. This is probably due to the lack of under- and over-conjugated species in the preparation, which can lead to ineffective or overly toxic molecules, respectively. Additionally, the non-cleavable linker used on the anti-CD22 ADC-
Maytansine payloads were resistant to efflux by MDR1 and did not result in off-target or bystander killing. Taken together, these features contributed to the observed efficacy and safety of anti-CD22 ADCs in preclinical studies.

Materials and Methods General All animal studies were approved by the Institutional Animal Care and Use Committee (Institutional Animal Care and Use Committee).
Imal Care and Use Committee) guidelines, C
Harles River Laboratories, Aragen Bioscience
was performed at nce or Covance Laboratories. A mouse anti-maytansine antibody was produced by ProMab and validated in-house. Rabbit anti-AF488
Antibodies were purchased from Life Technologies. Horseradish peroxidase (HRP)-conjugated secondary antibody was from Jackson Immunoresearch. Antibodies used for pharmacodynamic studies were from BD Pharmingen. Cell lines were obtained from ATCC and DSMZ cell banks, where they were authenticated by morphology, karyotyping and PCR-based approaches.

Cloning, Expression and Purification of Tagged Antibodies Antibodies were produced using standard cloning and purification techniques and GPEx® expression technology.

Bioconjugation, Purification and HPLC Analysis Drake et al., Bioconjugate Chem. , 2014, 25, 1331-
ADCs were fabricated and characterized as described in 41.

Generation of MDR1+ Cell Lines MDR1 (ABCB1) cDNA was obtained from Sino Biological and cloned into a pEF plasmid containing the hygromycin selectable marker. Ramos (ATCC CRL-1923) and WSU-DLCL2 (DSM
Z ACC 575) AMAXA Nucle to electroporate cells
An ofector™ device was used. Hygromycin (Invitrogen 1
0687010), the pool was enriched with paclitaxel treatment (25 nM for up to 10 days) to further select for cells containing functional MDR1. 10
% fetal bovine serum (FBS) and 1×GlutaMax (Gibco 35050-0
79) and maintained under hygromycin selection in RPMI (Gibco 21870-092) supplemented with .

In Vitro Cytotoxicity Assay Cell lines were plated in 96-well plates (Costar 3610) at a density of 5×10 ⁴ cells/well in 100 μL of growth medium and allowed to settle for 5 hours. Serial dilutions of test samples were made in RPMI at 6-fold the final concentration and 20 μL was added to the cells. 37°C,
After 5 days of incubation at 5% _CO2 , Promega CellTiter 9
6® AQueous One Solution Cell Proliferation Assay (G358
1) was used to measure viability according to the manufacturer's instructions. GI ₅₀ curves were calculated in GraphPad Prism using the drug-to-antibody ratio (DAR) values of the ADCs and doses were normalized to payload concentration.

Xenograft Studies Female CB17 ICR SCID mice received WSU-DLCL2 in 50% Matrigel
or Ramos cells were inoculated subcutaneously. Tumors were measured twice weekly with the formula:

Tumor volume was estimated by (where w=tumor width and l=tumor length). Once tumors reached the desired mean volume, animals were randomized into groups of 8-12 mice and dosed to them as described below. Animals were euthanized at the end of the study or when tumors reached 2000 mm ³ .

Rat Toxicity Study and Toxicokinetic (TK) Analysis
Or a single intravenous dose of 60 mg/kg anti-CD22 ADC was administered (5 animals/group).
Animals were observed for 12 days after dosing. Day 0, Day 1, Day 4, Day 8 and Day 11
Body weight was recorded daily. Blood was collected from all animals at 8 hours and at days 5, 9 and 12 for toxicokinetic analysis (all time points) and clinical chemistry and hematology analysis (days 5 and 12). used. Toxicokinetic analysis was performed by ELISA using the same conditions and reagents as described for pharmacokinetic analysis.

Non-Human Primate Toxicity and TK Studies Cynomolgus monkeys (2/sex/group) received 10, 30 or 60 mg/kg anti-CD22 AD
Two doses of C were administered (every 21 days) followed by a 21 day observation period. Prior to dosing on Day 1 and on Days 8, 15, 22 (pre-dose), 29, 36 and 42
Body weight was assessed daily. Blood was drawn for toxicokinetic, clinical chemistry and hematology analyses, according to the schedule shown in Table 2. Toxicokinetic analysis was performed by ELISA using the same conditions and reagents as described for pharmacokinetic analysis. However, in this case the CD22-His protein was used as a capture reagent for total antibody and total ADC measurements.

Pharmacodynamic Studies in Non-Human Primates Whole blood samples from cynomolgus monkeys enrolled in the anti-CD22 ADC toxicity study were analyzed by flow cytometry to assess CD3+, CD20+ and CD3-/CD20- leukocyte populations. Briefly, 100 μL aliquots of whole blood were added with either fluorescein- and phycoerythrin-conjugated isotype control antibodies or fluorescein-conjugated anti-CD20 and phycoerythrin-conjugated anti-CD3 antibodies and incubated on ice for 30 minutes. Red blood cells were then treated with ammonium chloride solution (Stem Cell Technolo
gies) and the cells were washed twice with phosphate-buffered saline + 1% FBS. Labeled cells were analyzed by FACSCanto™ running FACSDiva™ software
Analysis was performed by flow cytometry on the instrument.

Pharmacokinetic (PK) Study Design In the mouse study, animals used in the Ramos xenograft experiments were sampled in three groups at various time points beginning 1 hour after the first dose and continuing over the observation period. In the rat study, male Sprague-Dawley rats (3 per group) received a 3 mg/kg bolus of ADC intravenously. 1 hour, 8 hours and 24 hours after dosing
Plasma was collected at time points and 2, 4, 6, 8, 10, 14 and 21 days. Plasma samples were stored at -80°C until use.

Analysis of PK and TK samples .
Quantitation was by ELISA as illustrated in FIG. For total antibodies, conjugates were captured with an anti-human IgG-specific antibody and detected with an HRP-conjugated anti-human Fc-specific antibody. For all ADCs, conjugates were captured with an anti-human Fab-specific antibody and detected with a mouse anti-maytansine primary antibody followed by an HRP-conjugated anti-mouse IgG subclass 1-specific secondary antibody. For all conjugates, conjugates were captured with an anti-maytansine antibody and detected with an HRP-conjugated anti-human Fc-specific antibody. Ultra TMB One-
Bound secondary antibody was detected using Step ELISA substrate (Thermo Fisher). After quenching with sulfuric acid, M with SoftMax Pro software
Signals were read by acquiring absorbance at 450 nm on an olecular Devices Spectra Max M5 plate reader. GraphPad Pri
Data were analyzed using sm and Microsoft Excel software.

Indirect ELISA CD22 Antigen Binding Maxisorp 96-well plates (Nunc) were coated with 1 μg/mL human CD22-His (Sino Biological) in PBS overnight at 4°C. Plates were blocked with casein buffer (ThermoFisher) followed by anti-CD
22 wild-type antibodies and ADCs were plated in 11 serial two-fold dilutions starting at 200 ng/mL. The plates were incubated at room temperature for 2 hours with shaking. After washing with phosphate-buffered saline (PBS) 0.1% Tween-20,
Bound analyte was detected with a donkey anti-human Fcγ-specific horseradish peroxidase (HRP)-conjugated secondary antibody. Signals were visualized with Ultra TMB (Pierce) and 2N
_Quenched with _H2SO4 . Molecular Devices SpectraMa
Measure absorbance at 450 nm using a x M5 plate reader and GraphPad
Data were analyzed using Prism.

Anti-CD22 ADC Mediated CD22 Internalization in CD22+ NHL Cell Lines Ramos, Granta-519 and WSU-DLCL2 cells (1e6/test) were treated in labeling buffer only [PBS + 1% fetal bovine serum (FBS)] or anti-CD
Incubated in labeling buffer containing 22 ADC (1 μg/test).
Samples were placed at 4 or 37°C for 2 hours. Cells were then treated with fluorescein-labeled anti-CD2
2 on ice for 20 minutes. After two washes in labeling buffer, FA
Cells were analyzed by flow cytometry on a FACSCanto™ instrument running CSDiva™ software. Differences in fluorescence between cells at 4° C. and 37° C.±ADC were interpreted as anti-CD22 ADC-mediated internalization.

Cross-reactivity studies in cynomolgus monkey and human tissues.
a Biosystems Inc. (Richmond, Calif.) performed tissue cross-reactivity studies. A tissue microarray containing skin, heart, lung, kidney, liver, pancreas, stomach, small intestine, large intestine and spleen (positive control) was used. Primary antibodies were detected using horseradish peroxidase-conjugated streptavidin followed by visualization with DAB substrate.

Synthesis of HIPS-4AP-maytansine linker payload

(9H-fluoren-9-yl)methyl 1,2-dimethylhydrazine-1-carboxylate (2)
MeNHNHMe.2HCl (1) (5.0 g, 37.6 mmol) was dissolved in CH ₃ CN (8
0 mL). Et ₃ N (22 mL, 158 mmol) was added and the resulting precipitate was removed by filtration. FmocCl (0.49 g, 18.9 g) was added to the remaining solution of MeNHNHMe.
mmol, 0.5 eq.) was added dropwise at −20° C. over 2.5 hours. The reaction mixture was then diluted with EtOAc, washed with _H2O , _brine , dried over _Na2SO4 and concentrated in vacuo. The residue was flash chromatographed on silica (hexane:EtOAc=
3:2) to give 3.6 g (34%) of compound 2.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.75-7.37 (m, 8H), 4
. 48 (br s, 2H), 4.27 (t, J=6.0Hz, 1H), 3.05 (s, 3
H), 2.55 (br s, 3H).

2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-indole (
4)
Indole-2-methanol, 3 (1.581 g, 10.5 g) was added to an oven-dried flask.
74 mmol), TBSCl (1.789 g, 11.87 mmol) and imidazole (2.197 g, 32.27 mmol) were charged and the mixture was treated with CH ₂ Cl ₂ (40 mL,
anhydrous). After 16 hours, the reaction mixture was concentrated to give an orange residue. The crude mixture was taken in Et ₂ O (50 mL) and washed with aqueous AcOH (5% v/v, 3 x 50 mL) and brine (25 mL). The combined organic layers were dried over Na ₂ SO ₄ and concentrated to give 2.789 g (99%) of compound 4 as a crystalline solid, which was used without further purification.

¹ H NMR (500 MHz, CDCl ₃ ) δ 8.29 (s, 1H), 7.57 (d
, J=7.7 Hz, 1 H), 7.37 (dd, J=8.1, 0.6 Hz, 1 H), 7.1
9-7.14 (m, 1H), 7.12-7.07 (m, 1H), 6.32 (d, J=1.
0Hz, 1H), 4.89 (s, 2H), 0.95 (s, 9H), 0.12 (s, 6H)
.

¹³ C NMR (101 MHz, CDCl ₃ ) δ 138.3, 136.0, 128.
6, 121.7, 120.5, 119.8, 110.9, 99.0, 59.4, 26.1
, 18.5, -5.2.

HRMS (ESI) _calcd for _C15H24NOSi [M+H] ⁺ : 262.1
627; Found: 262.1625.

methyl 3-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H
- indol-1-yl)propanoate (6)
Methyl acrylate, 5 (4.80 mL, 53.3 mmol) was added to a solution of indole 4 (2.789 μg, 10.67 mmol) in CH ₃ CN (25 mL) followed by 1,8.
- diazabicyclo[5.4.0]undec-7-ene (800 μL, 5.35 mmol)
was added and the resulting mixture was brought to reflux. After 18 hours, the solution was cooled and concentrated to give an orange oil, which was purified by silica gel chromatography (9:1 hexanes:EtOAc) to give 3.543 g (96%) of compound 6 as a colorless oil. obtained as

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.58 (d, J=7.8 Hz, 1H
), 7.34 (d, J = 8.2 Hz, 1H), 7.23-7.18 (m, 1H), 7.1
2-7.07 (m, 1H), 6.38 (s, 1H), 4.84 (s, 2H), 4.54-
4.49 (m, 2H), 2.89-2.84 (m, 2H), 0.91 (s, 9H), 0.
10(s, 6H).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 172.0, 138.5, 137.
1, 127.7, 122.0, 121.0, 119.8, 109.3, 101.8, 58
. 2, 51.9, 39.5, 34.6, 26.0, 18.4, -5.2.

HRMS ₍ ESI) _calcd for _C19H30NO3Si [M+H] ⁺ : 348.
1995; Found: 348.1996.

Methyl 3-(2-(hydroxymethyl)-1H-indol-1-yl)propanoate (7)
To a solution of compound 6 (1.283 g, 3.692 mmol) in THF (20 mL) at 0° C. was added a 1.0 M solution of tetrabutylammonium fluoride in THF (3.90 mL, 3.90 mL).
mmol) was added. After 15 min, the reaction mixture was diluted with Et ₂ O (20 mL) and NaHC.
Wash with O ₃ (saturated aqueous solution, 3×20 mL) and concentrate to give a pale green oil. The oil was purified by silica gel chromatography (2:1 hexanes:EtOAc) to give 82
Obtained 2 mg (95%) of 7 as a white crystalline solid.

¹ H NMR (500 MHz, CDCl ₃ ) δ 7.60 (d, J=7.8 Hz, 1H
), 7.34 (dd, J = 8.2, 0.4 Hz, 1H), 7.27-7.23 (m, 1H
), 7.16-7.11 (m, 1H), 6.44 (s, 1H), 4.77 (s, 2H),
4.49 (t, J=7.3 Hz, 2H), 3.66 (s, 3H), 2.87 (t, J=7
. 3Hz, 2H), 2.64(s, 1H).

¹³ C NMR (126 MHz, CDCl ₃ ) δ 172.3, 138.5, 137.
0, 127.6, 122.2, 121.1, 119.9, 109.3, 102.3, 57
. 1, 52.0, 39.1, 34.3.

HRMS ₍ ESI) _calcd for _C13H15NNaO3 [M+Na] ⁺ : 256
. 0950; Found: 256.0946.

Methyl 3-(2-formyl-1H-indol-1-yl)propanoate (8)
Dess-Martin periodinane (5.195 g, 12.25 mmol) in CH ₂ Cl
Suspended in a mixture of ₂ (20 mL) and pyridine (2.70 mL, 33.5 mmol). After 5 minutes, the resulting white suspension was treated with methyl 3-(2-(hydroxymethyl)-1H-indol-1-yl)propanoate (7; 2.611 g, 7 in CH ₂ Cl ₂ (10 mL).
11.19 mmol) to give a reddish brown suspension. After 1 hour, the reaction was quenched with sodium thiosulfate (10% aqueous solution, 5 mL) and NaHCO ₃ (saturated aqueous solution, 5 mL). The aqueous layer was extracted with CH2Cl2 ( ₃ x ₂₀ mL). The combined extracts _were washed with Na2
Dried over _SO4 , filtered and concentrated to give a brown oil. Purification by silica gel chromatography (5-50% EtOAc in hexanes) gave 2.165 g (84%) of compound 8 as a colorless oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.87 (s, 1H), 7.73 (d
t, J=8.1, 1.0 Hz, 1 H), 7.51 (dd, J=8.6, 0.9 Hz, 1 H
), 7.45-7.40 (m, 1H), 7.29 (d, J=0.9Hz, 1H), 7.1
8 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 4.84 (t, J = 7.2 Hz
, 2H), 3.62 (s, 3H), 2.83 (t, J=7.2 Hz, 2H).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 182.52, 171.75, 14
0.12, 135.10, 127.20, 126.39, 123.46, 121.18,
118.55, 110.62, 51.83, 40.56, 34.97.

HRMS ₍ ESI) _calcd for _C13H13NO3Na [M+Na] ⁺ : 254
. 0793; Found: 254.0786.

3-(2-formyl-1H-indol-1-yl)propanoic acid (9)
Indole 8 (2.369 g, 10.24 mmol) dissolved in dioxane (100 mL)
LiOH (4M aqueous solution, 7.68 mL, 30.73 mmol) was added to the solution of l). A thick white precipitate formed gradually over several hours. After 21 hours, HCl (1 M aqueous solution, 30
mL) was added dropwise to obtain a pH 4 solution. The solution was concentrated and the resulting light brown oil was treated with EtO.
Dissolve in Ac (50 mL) and wash with water (2 x 50 mL) and brine (20 mL). The organic layer was dried over _Na2SO4 , _filtered and concentrated to give an orange solid. Silica gel chromatography (10-50% EtOAc in hexanes containing 0.1% acetic acid
Purification by c) gave 1.994 g (84%) of compound 9 as a pale yellow solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.89 (s, 1H), 7.76 (d
t, J = 8.1, 0.9 Hz, 1 H), 7.53 (dd, J = 8.6, 0.9 Hz, 1 H
), 7.48-7.43 (m, 1H), 7.33 (d, J=0.8Hz, 1H), 7.2
1 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 4.85 (t, J = 7.2 Hz
, 2H), 2.91 (t, J=7.2 Hz, 2H).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 182.65, 176.96, 14
0.12, 135.02, 127.33, 126.42, 123.53, 121.27,
118.76, 110.55, 40.19, 34.82.

HRMS (ESI) _calcd for _C12H10NO3MH ] ^- : _216.06
66; Found: 216.0665.

3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1
, 2-dimethylhydrazinyl)methyl)-1H-indol-1-yl)propanoic acid (1
0)
Compound 9 (1.193 g, 5.492 g) in 1,2-dichloroethane (anhydrous, 25 mL)
mmol) and (9H-fluoren-9-yl)methyl-1,2-dimethyl hydrazinecarboxylate, 2 (2.147 g, 7.604 mmol) was added with sodium triacetoxyborohydride (1.273 g, 6.006 mmol). ) was added. The resulting yellow suspension was stirred for 2 hours, then quenched with NaHCO ₃ (saturated aqueous solution, 10 mL) and then HCl (1M aqueous solution) was added to pH4. _The organic layer was separated and the aqueous layer was _treated with CH2Cl2.
(5 x 10 mL). The combined organic extracts _were dried over _Na2SO4 , filtered and concentrated to give an orange oil. C18 silica gel chromatography (20-9 in water
Purification with 0% CH ₃ CN) gave 1.656 g (62%) of compound 10 as a waxy pink solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.76 (d, J = 7.4 Hz, 2H
), 7.70-7.47 (br m, 3H), 7.42-7.16 (br m, 6H),
7.12-7.05 (m, 1H), 6.37 (s, 0.6H), 6.05 (s, 0.4H)
), 4.75-4.30 (br m, 4H), 4.23 (m, 1H), 4.10 (br
s, 1H), 3.55 (br d, 1H), 3.11-2.69 (m, 5H), 2.57
(br s, 2H), 2.09 (br s, 1H).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 174.90, 155.65, 14
3.81, 141.42, 136.98, 134.64, 127.75, 127.48,
127.12, 124.92, 122.00, 120.73, 120.01, 119.7
5, 109.19, 103.74, 67.33, 66.80, 51.39, 47.30,
39.58, 39.32, 35.23, 32.10.

HRMS ₍ ESI) _calc'd for _C29H30N3O4 [M+H] ⁺ : _484.2 .
236; Found: 484.2222.

(9H-fluoren-9-yl)methyl 1,2-dimethyl-2-((1-(3-oxo-3-(perfluorophenoxy)propyl)-1H-indol-2-yl)methyl)hydrazine-1- Carboxylate (RED-004)
Compound 10 (5.006 g) was added to a dry 100 mL two-necked round bottom flask containing a dry stir bar.
, 10.4 mmol) was added. Anhydrous EtOAc (40 mL) was added via syringe and the solution was stirred at 20° C. for 5 minutes to give a clear pale yellow-green solution. The solution was cooled to 0° C. in an ice-water bath and treated with pentafluorophenol (2098.8 mg, 11.4 mg) in 3 mL of anhydrous EtOAc.
mmol) was added dropwise. The solution was stirred at 0° C. for 5 minutes. DC in 7 mL anhydrous EtOAc
C (2348.0 mg, 11.4 mmol) was slowly added dropwise via syringe. The solution was stirred at 0°C for 5 minutes, then removed from the bath and warmed to 20°C. The reaction was stirred for 2 hours, cooled to 0° C. and filtered to give a clear pale yellow-green solution. The solution was diluted with 50 mL EtOAc, washed with ₂ x 25 mL _H2O , 1 x 25 mL 5M NaCl and dried over _Na2SO4 . The solution was filtered, evaporated and dried under high vacuum to yield 6552.5 mg (97%
) as a green-white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 780 (d, J=7.2 Hz, 2 H)
, 7.58(m, 3H), 7.45-7.22(m, 6H), 7.14(dd(appt
. t), J=7.4 Hz, 1 H), 6.42 & 6.10 (2 br s, 1 H), 4.7
4(dd(appt.t), J=5.4Hz, 2H), 3.65-3.18(br, 3H
), 3.08 & 2.65 (2 br s, 3H), 2.88 (s, 3H).

(S)-3,4-dimethyloxazolidine-2,5-dione (RED-194)
N-Boc-Ala-OH (11) (0.005) in methylene chloride (25 ml) at 0°C
mol) under nitrogen was added 1.2 equivalents of phosphorus trichloride. The reaction mixture is stirred at 0° C. for 2 hours, the solvent is removed under reduced pressure and the residue is washed with carbon tetrachloride (3×20 ml) to give RED-1.
94 was obtained.

(1 ⁴ S, 1 ⁶ S, 3 ² R, 3 ³ R, 2R, 4S, 10E, 12E, 14R) -8 ⁶ -
Chloro-1 ⁴ -hydroxy-8 ⁵ ,14-dimethoxy-3 ³ ,2,7,10-tetramethyl-1 ² ,6-dioxo-7-aza-1(6,4)-oxazinana-3(2, 3)-oxirana-8(1,3)-benzeneacyclotetradecaphane-10,12-diene-4-
yl methyl-L-alaninate (RED-062)
Maytansinol (RED-063) (4.53 g, 8 mmol) was dissolved in anhydrous DMF (11
mL) to give a clear, colorless solution, which was transferred to a dry two-necked round bottom flask under _N2 . Anhydrous THF (44 mL) was added followed by DIPEA (8.4 mL, 48 mmol). A solution of RED-194 (5.4 g, 42 mmol) was added to give a clear colorless solution. Add dried micronised Zn(OTf) ₂ (8.7 g, 24 mmol) to the stirring solution,
The reaction mixture was stirred at 20° C. for 2 days. 70 mL of 1.2 M _NaHCO3 and 70 mL
The reaction was quenched by adding a solution of L in EtOAc. The resulting mixture produced a white precipitate upon stirring which was removed by filtration. The filtrate was diluted with EtOAc (5 x 70 m
L), dried (Na ₂ SO ₄ ) and concentrated to give a red-orange oil. Change this to CH
₂ Cl ₂ (15 mL) and added to the Biotage system (2 x Biotage Ult
ra Adsorbed on 10 g sample, 2×Bi in 0-20% gradient of MeOH in CH ₂ Cl ₂
Purification on otage Ultra 100 g cartridge) to give 4.38
g of RED-062 was obtained as a pale pink solid (95% de, 93.7% desired diastereomer).

tert-butyl 4-oxopiperidine-1-carboxylate (13)
Piperidin-4-one hydrochloride monohydrate (
12) (1.53 g, 10 mmol), di-tert-butyl dicarbonate (2.39
g, 11 mmol), sodium carbonate (1.22 g, 11.5 mmol), dioxane (
10 mL) and water (1 mL) were added. The reaction mixture was stirred at room temperature for 1 hour. The mixture was diluted with water (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers _were washed with brine, dried over _Na2SO4 , filtered and concentrated under reduced pressure. The resulting material was dried in vacuo to give 1.74 g (87%) of compound 13 as a white solid.

¹ H NMR (CDCl ₃ ) δ 3.73 (t, 4H, J=6.0), 2.46 (t,
4H, J=6.0), 1.51 (s, 9H).

MS ₍ ESI) m/z: _calc'd for [M+H] ⁺ _C10H18NO3 : 200.
3; Found value: 200.2.

tert-butyl 4-((2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)amino)piperidine-1-carboxylate (14)
Compound 13 (399 mg, 2 mmo) was added to a dry scintillation vial containing a magnetic stir bar.
l), H ₂ N-PEG ₂ -COOt-Bu (550 mg, 2.4 mmol), 4 Angstrom molecular sieves (activated powder, 200 mg) and 1,2-dichloroethane (5 mL) were added. The mixture was stirred at room temperature for 1 hour. Sodium triacetoxyborohydride (845 mg, 4 mmol) was added to the reaction mixture. The mixture was stirred at room temperature for 3 days. The resulting mixture was partitioned between EtOAc and saturated aqueous _NaHCO3 . The organic layer was washed with brine, dried over _Na2SO4 , filtered and concentrated under reduced pressure to give 850 mg of compound ₁₄ as a viscous oil.

MS (ESI) m/z: _calc'd for [M ₊ H] ⁺ _C21H41N2O6 : ₄₁₇ .
. 3; Found: 417.2.

13-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadecane-1
7-acid (RED-195)
Compound 14 (220 mg, 0.5 mL) was added to a dry scintillation vial containing a magnetic stir bar.
mol), succinic anhydride (55 mg, 0.55 mmol), 4-(dimethylamino)pyridine (5 mg, 0.04 mmol) and dichloromethane (3 mL) were added. The mixture was stirred at room temperature for 24 hours. The reaction mixture was flash chromatographed (50-100%
EtOAc/hexanes) to give 117 mg of compound RED-195.
was obtained as a clear oil, which was carried forward without further characterization.

MS (ESI) m/z: _calc'd for [M ₊ H] ⁺ _{C25H45N2O 9} : 517.
. 6; Found: 517.5.

17-(tert-butyl) 1-((1 ⁴ S, 1 ⁶ S, 3 ³ S, 2R, 4S, 10E
, 12E, 14R)-8 ⁶ -chloro-1 ⁴ -hydroxy-8 ⁵ ,14-dimethoxy-3 ³
,2,7,10-tetramethyl-1 ² ,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzeneacyclotetra Decafane-10,12-dien-4-yl) (2S)-8-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2,3-dimethyl-4,7-dioxo-11,14 -dioxa-3,8-diazaheptadecanedioate (RED-196)
RED-195 (445 mg, 0.05 mg, 0.05 mg) was added to a dry scintillation vial containing a magnetic stir bar.
86 mmol), HATU (320 mg, 0.84 mmol), DIPEA (311 mg
, 2.42 mmol) and dichloromethane (6 mL) were added. The reaction mixture was allowed to stand at room temperature for 5
Stir for a minute. The resulting solution was added to RED-062 (516 mg, 0.79 mmol) and the reaction mixture was stirred at room temperature for an additional 30 minutes. The reaction mixture was directly purified by flash chromatography (eluting with 3-10% MeOH/DCM) to give 820 mg (90%) of R
ED-196 was obtained as a pale brown solid.

MS _{( ESI} ) m/z: _calcd for [M+H] ^< +> _{C57H87ClN5O17} :
1148.6; Found: 1148.8.

(2S)-1-((1 ⁴ S, 1 ⁶ S, 3 ³ S, 2R, 4S, 10E, 12E, 14R)
-8 ⁶ -chloro-1 ⁴ -hydroxy-8 ⁵ ,14-dimethoxy-3 ³ ,2,7,10-tetramethyl-1 ² ,6-dioxo-7-aza-1(6,4)-oxazinana- 3 (2,3
)-oxirana-8(1,3)-benzeneacyclotetradecaphan-10,12-dien-4-yl)oxy)-2,3-dimethyl-1,4,7-trioxo-8-(piperidine- 4-yl)-11,14-dioxa-3,8-diazaheptadecan-17-acid (RE
D-197)
RED-196 (31 mg, 0.0
27 mmol) and dichloromethane (1 mL) were added. The solution was cooled to 0° C. and tin(IV) tetrachloride (1.0 M solution in dichloromethane, 0.3 mL, 0.3 mmol) was added. The reaction mixture was stirred at 0° C. for 1 hour. The reaction mixture was directly purified by C18 flash chromatography (eluting with 5-100% MeCN/water) to give 16 mg (60%) of R
ED-197 was obtained as a white solid (16 mg, 60% yield).

MS _{( ESI} ) m/z: _calcd for [M+H] ^< +> _{C48H71ClN5O15} :
992.5; Found: 992.6.

(2S)-8-(1-(3-(2-((2-((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-indole- 1-
yl)propanoyl)piperidin-4-yl)-1-(((1 ⁴ S, 1 ⁶ S, 3 ³ S, 2
R,4S,10E,12E,14R)-8 ⁶ -chloro-1 ⁴ -hydroxy-8 ⁵ ,14-
Dimethoxy-3 ³ ,2,7,10-tetramethyl-1 ² ,6-dioxo-7-aza-1 (
6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzeneacyclotetradecafane-10,12-dien-4-yl)oxy)-2,3-dimethyl-1 ，
4,7-trioxo-11,14-dioxa-3,8-diazaheptadecan-17-acid (
RED-198)
RED-197 (16 mg, 0.00
16 mmol), (9H-fluoren-9-yl)methyl 1,2-dimethyl-2-((
1-(3-oxo-3-(perfluorophenoxy)propyl)-1H-indole-2
-yl)methyl)hydrazine-1-carboxylate (12) (13 mg, 0.02 mm)
ol), a mixture of DIPEA (8 μL, 0.05 mmol) and DMF (1 mL) was added. The solution was stirred at room temperature for 18 hours. The reaction mixture was directly purified by C18 flash chromatography (eluting with 5-100% MeCN/water) to give 18 mg (77%) of R
ED-198 was obtained as a white solid.

MS _{( ESI} ) m/z: _calcd for [M+H] ⁺ _{C77H98ClN8O18} :
1457.7; Found: 1457.9.

(2S)-1-(((1 ⁴ S, 1 ⁶ S, 3 ³ S, 2R, 4S, 10E, 12E, 14R
)-8 ⁶ -chloro-1 ⁴ -hydroxy-8 ⁵ ,14-dimethoxy-3 ³ ,2,7,10-
Tetramethyl-1 2,6-dioxo-7-aza-1(6,4)-oxazinana-3( ² ,
3)-oxirana-8(1,3)-benzeneacyclotetradecaphan-10,12-dien-4-yl)oxy)-8-(1-(3-(2-((1,2-dimethyl) hydrazinyl)methyl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)-2
,3-dimethyl-1,4,7-trioxo-11,14-dioxa-3,8-diazaheptadecan-17-acid (RED-106)
RED-197 (18 mg, 0.00
12 mmol), piperidine (20 μL, 0.02 mmol) and DMF (1 mL) were added. The solution was stirred at room temperature for 20 minutes. The reaction mixture was purified directly by C18 flash chromatography (eluting with 1-60% MeCN/water) to give 15 mg (98%) of compound RED-106 as a white solid.

MS _{( ESI} ) m/z: _calcd for [M+H] ^< +> _{C62H88ClN8O16} :
1235.6; Found: 1236.0.

Results and Discussion Anti-CD22 ADC Production and Initial Characterization The anti-CD22 antibody used (CAT-02) was a humanized variant of the RFB4 antibody. 1
. C-terminal tagged anti-CD22 using a GPEx® clonal cell line showing a bioreactor titer of 6 g/L and 97% conversion of cysteine to formylglycine
Antibodies were produced. A HIPS-4AP-maytansine linker payload was synthesized (described above) and conjugated to the aldehyde-tagged antibody. The resulting ADC was analyzed by size exclusion chromatography to assess monomer content (99.2%) and to assess drug-to-antibody ratio (DAR), which was 1.8. to the hydrophobic interaction (HI
C) and characterized by reverse phase (PLRP) chromatography (Fig. 11). Human C
For affinity to the D22 protein and internalization on CD22+ cells, the ADC was tested against wild-type (untagged) anti-CD22 using ELISA-based (Figure 12) and flow cytometry-based methods (Figure 13), respectively. compared to antibodies. For both functional measures, the ADC performed as well as the wild-type antibody, indicating that conjugation had no effect on these parameters.

Anti-CD22 ADCs are not substrates of MDR1 and do not promote off-target or bystander killing The efficacy of anti-CD22 ADCs was tested in vitro against Ramos and WSU-DLCL2 HNL tumor cell lines. CAT-02 conjugated to maytansine via a cleavable valine-citrulline dipeptide linker
The activity was compared to that of related ADCs prepared using anti-CD22 antibodies. Both ADCs exhibited sub-nanomolar activity against wild-type Ramos and WSU-DLCL2 cells (Fig. 1).
4 panel A and panel C). In mutant cells engineered to express the xenobiotic efflux pump MDR1, only the anti-CD22 ADCs of the present disclosure retained their original potency (FIG. 14 panels B and D). In contrast, free maytansine exhibited ~10-fold lower potency and ADCs containing cleaving maytansines were virtually devoid of activity. In control experiments, co-treatment of WSU-DLCL2 cells with the MDR1 inhibitor cyclosporine had no effect on wild-type cells, whereas MDR1
It restored the original potency of free maytansine and cleaving ADC in + cells (Figure 14 panels E and F). Taken together, these results indicated that the active metabolites of the anti-CD22 ADCs of this disclosure are not substrates for MDR1 efflux. In a relevant in vitro cytotoxicity study, the anti-CD22 ADCs of the disclosure had no effect on the antigen-negative cell line NCI-N87 (Figure 15). This indicates that it exhibited no off-target activity over the 5-day cell culture period. Furthermore, an anti-HER2-based ADC conjugated to the HIPS-4AP-maytansine linker payload did not result in bystander killing of antigen-negative cells in co-culture with antigen-positive cells (FIG. 16).
This is the same as for the anti-HER2 ADC conjugates of the present disclosure.
2 ADC's active metabolites also do not result in bystander killing.

Anti-CD22 ADCs were Effective Against NHL Xenograft Models The in vivo efficacy of anti-CD22 ADCs was tested in WSU-DLCL2 and Ramos xenograft models (FIG. 17), which were at relatively higher and lower doses, respectively. CD22
expressed) (Fig. 18). In single-dose (single-dose) studies, WSU
- DLCL2 tumor-bearing mice were administered 10 mg/kg anti-CD22 ADC or vehicle control. Dosing began when tumors averaged 118 mm ³ . Of the animals that received ADC, 25% (2 of 8) showed a partial response and their tumors were 31
Regressed to 4 mm ³ by day. The anti-CD22 ADC-treated group and vehicle control group were grouped in the third
By day 1, they had mean tumor volumes of 415 and 1783 mm ³ , respectively. In a multiple dose (multidose) study, mice bearing WSU-DLCL2 xenografts were then treated with 10 mg/kg anti-CD22 ADC or vehicle control (every 4 days for a total of 4 doses).
processed with Dosing began when tumors averaged 262 mm ³ . Of the animals dosed with ADC, 75% (6 out of 8) showed a complete response and 38% (3 out of 8) of these were at the end of the study (day 59) (43 days after the last dose). continued it until In contrast, the vehicle control group reached a mean tumor volume of 2191 mm ³ by day 17. Finally, in a multiple dose study, mice bearing Ramos xenografts were treated with 5 or 10 mg/kg
of anti-CD22 ADC or vehicle control (every 4 days for a total of 4 doses). Dosing began when tumors averaged 246 mm ³ . 5 or 10 mg/kg, as expected
A dose effect was observed indicating tumor growth retardation of 63% or 87%, respectively, in the dose-treated groups. Notably, the median time to endpoint was higher than the vehicle control group at 5 mg/day.
12, 19 and 22 days for the kg and 10 mg/kg dose groups, respectively. In all three studies, no effect on body weight of mice in the anti-CD22 ADC-treated group was observed (Figure 19).

Anti-CD22 ADC was well tolerated up to 60 mg/kg in rats and cynomolgus monkeys.
Administration of DCs provided information on off-target toxicity and safety of the linker payload. As noted above, no treatment effects on body weight or clinical findings were observed in mouse xenograft studies. In an exploratory rat toxicity study (Figure 20), animals (5 per group) were administered a single intravenous dose of 6, 20, 40 or 60 mg/kg of anti-CD22 ADC and observed for 12 days post-dosing. All animals survived until the end of the study. 60mg/
Animals dosed with kg showed a 10% reduction in body weight compared to the vehicle control group. Clinical chemistry changes compatible with minimal to mild hepatobiliary injury were found on day 5 in animals dosed at 40 mg/kg and above, including alanine aminotransferase (ALT
), aspartate transaminase (AST) and alkaline phosphatase (AL
P) activity increase. Most changes were reversed by day 12. With regard to hematology, moderate to markedly decreased platelet counts were found on day 5 in animals dosed at 40 mg/kg and above, with a complete reversal by day 12. Inflammation-matched changes were found on days 5 and 12 in animals dosed at 40 mg/kg, which included slightly to moderately increased neutrophil and monocyte counts, slightly increased Globulin concentrations and decreased albumin:globulin ratios were included.

The anti-CD22 ADC bound to cynomolgus monkey CD22 (Figure 21) and showed a similar tissue cross-reactivity profile in monkeys compared to humans (Figure 22). Cynomolgus monkeys therefore represented an appropriate model for testing both on- and off-target toxicity of this ADC. In an exploratory repeated-dose study, monkeys (2/sex/group)
were administered 10, 30 or 60 mg/kg of anti-CD22 ADC once every 3 weeks for a total of 2 doses, followed by a 21-day observation period. All animals survived until the end of the study. No anti-CD22 ADC-related changes in clinical findings, body weight or food intake occurred. Clinicopathologic changes occurred primarily in animals dosed at 30 mg/kg and above, consistent with minimal liver injury, elevated platelet consumption and/or loss and inflammation (Figure 23). These changes were similar after 30 and 60 mg/kg and first and second doses,
The magnitude was not expected to be associated with microscopic changes or clinical efficacy. 30mg/
Changes compatible with minimal liver injury in animals dosed at ≥4 kg were observed on days 21 and 4
It consisted of elevated ALT, AST and ALP activity that was partially reversed by day 2. Slightly to moderately decreased platelet counts observed within 1 week of dosing were 21
By day 42 and day 42 there was almost a reversal. Inflammation-matched changes were minimal to moderately increased neutrophil and monocyte counts, slightly to moderately increased globulin levels and minimally decreased albumin levels.

Administration of anti-CD22 ADCs resulted in B cell depletion in cynomolgus monkeys Peripheral blood in samples collected from cynomolgus monkeys enrolled in a repeated-dose toxicity study to assess the pharmacodynamic effects of anti-CD22 ADCs in cross-reactive species Monocyte populations were monitored. Specifically, B cells (CD20+), T cells (CD3+) and NK observed in animals prior to dosing and at days 7, 14, 28 and 35
Flow cytometry was used to find the ratio of cells (CD20-/CD3-) (Fig. 5). In anti-CD22 ADC-treated animals prior to dosing, B cells averaged 11
. contained 6%. This value decreased to an average of 3.8% by day 35. This corresponds to a mean reduction of 68% in the measured B cell population compared to baseline levels (Fig. 24).
). B cell depletion was similar in all dose groups from 10 to 60 mg/kg, indicating that the lowest dose was sufficient to produce the effect. In contrast, B cells in vehicle control treated animals and T cells and NK cells in all groups (not shown) changed little over the course of treatment. These results demonstrated that anti-CD22 ADCs can selectively cause depletion of cynomolgus monkey CD22+ cells in vivo without adverse off-target toxicity.

Pharmacokinetics and toxicokinetics of anti-CD22 ADCs in mice, rats and cynomolgus monkeys To assess the in vivo stability of anti-CD22 ADCs, pharmacokinetics in rats (P
K) was tested. Concentrations of total antibody, total ADC and total conjugate in the peripheral blood of animals (3/group) were monitored for 21 days after administration of a single dose of 3 mg/kg of anti-CD22 ADC (Table 2 and Figure 25). ). As shown in Figure 10, all ADC and all conjugate assays used DAR sensitive and DAR insensitive measurements, respectively. The PK parameters obtained for all three analytes were similar, indicating that the conjugate was largely stable in circulation. For example, the elimination half-life of whole antibody, whole ADC and whole conjugate are 9.48, 6.13, respectively.
and 7.22 days.

Next, anti-CD
The concentration of 22 ADC analyte was measured over time. The purpose of this analysis was to determine the total ADC exposure levels achieved at effective doses in xenograft trials (Figure 26). For this criterion, 10 mg/kg x 4 doses over 22 days resulted in 87% tumor growth delay in the Ramos model, and 10 mg/kg x 4 doses over 28 days.
Recall that the dose produced a complete response (no palpable tumor remaining) in 75% of animals in the WSU-DLCL2 model. 10 mg/kg in the mouse
Mean area under the concentration vs. time curve from time 0 to infinity for ×4 doses (AUC _0-inf
) was 2530±131 (SD) days·μg/mL.

Finally, anti-CD22 ADC analyte concentrations in toxicokinetic plasma samples from animals dosed in the rat and cynomolgus monkey toxicity studies described above were assessed (Figure 26). The purpose of these analyzes was to determine total ADC exposure levels achieved at doses that correlated with the presence or absence of observed toxicity. For the rat study, C _max and A
UC _0-inf values were generally proportional to dose. Mean AUC _0-in at 60 mg/kg dose
_f was 5201±273 days·μg/mL. For the monkey study, C _max and AUC _0-inf values were generally proportional to dose. Mean AUC for first 60 mg/kg dose
The _0-inf was 6140±667 days·μg/mL. The antibody bound to antigen in the cynomolgus monkey model, but clearance (not shown) was similar in all dose groups. This is low (10 mg
/kg) dose is sufficient, thus demonstrating that antigen-mediated clearance does not significantly affect the results of this study. This observation suggests that anti-CD22 A on B cell depletion
Consistent with pharmacodynamic effects of DC treatment, the magnitude was similar in all dose groups.

Conclusions A CD22-targeting ADC site-specifically conjugated to a maytansine payload was produced that is resistant to efflux by MDR1-expressing cells. The ADC has a D of 1.8
It has an AR and exhibits good biophysical properties, resulting in efficacy ranging from significant (87%) tumor growth delay to complete responses in vivo in two NHL xenograft models. This efficacy was achieved at exposure levels well below those associated with toxicity. Indeed, in repeated-dose cynomolgus monkey toxicity studies, no side effects were observed even at the highest dose of 60 mg/kg, indicating that higher doses can be used. anti-CD22
ADC had both efficacy and safety. As an additional benefit, the target antigen,
A number of building blocks, including parent antibodies and maytansine-based cytotoxic payloads, have been used in humans and have been well studied with regard to safety and toxicity. Based on cynomolgus monkeys, a rational model for predicting human pharmacokinetic and toxicity profiles,
The results of these studies indicated that anti-CD22 ADCs are therapeutically useful in NHL patients, such as those with refractory disease due to upregulation of MDR1.

Although the invention has been described with respect to particular embodiments thereof, various modifications can be made and equivalents substituted (substituted) without departing from the true spirit and scope of the invention.
It should be understood by those skilled in the art that In addition, individual circumstances, materials, compositions, methods,
Many modifications may be made to adapt the method steps or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims (7)

Use of a pharmaceutical composition in the manufacture of a medicament for delivering the pharmaceutical composition to a target site in a subject, the pharmaceutical composition comprising formula (I)

(wherein W2 is an anti ^- CD22 antibody comprising at least one modified amino acid residue)
The use above, including the conjugate of 2. The anti-CD22 antibody of claim 1, wherein said anti-CD22 antibody binds to an epitope within amino acids 1 to 846, amino acids 1 to 759, amino acids 1 to 751, or amino acids 1 to 670 of the CD22 amino acid sequence set forth in Figures 8A to 8C. Use as indicated. wherein said anti-CD22 antibody has formula (II)
X ¹ (FGly') X ² Z ²⁰ X ³ Z ³⁰ (II)
(In the formula,
FGly' is a modified amino acid residue of formula (I);
^Z20 is a proline or alanine residue;
Z ³⁰ is a basic amino acid or an aliphatic amino acid;
X ¹ may or may not be present, and if present may be ^any amino acid, but is present if said sequence is at the N-terminus of said conjugate; and X2 and X3 ^{are each} independently any amino acid),
Preferably,
a) said sequence is L(FGly')TPSR, or b) Z ³⁰ is selected from R, K, H, A, G, L, V, I and P;
X ¹ is selected from L, M, S and V; and X ² and X ³ are each independently selected from S, T, A, V, G and C;
Use according to claim 1 . Said modified amino acid residue is located at the C-terminus of the heavy chain constant region of the anti-CD22 antibody, preferably said heavy chain constant region has the formula (II):
X ¹ (FGly') X ² Z ²⁰ X ³ Z ³⁰ (II)
(In the formula,
FGly' is a modified amino acid residue of formula (I);
^Z20 is a proline or alanine residue;
Z ³⁰ is a basic amino acid or an aliphatic amino acid;
X ¹ may or may not be present, and if present may be ^any amino acid, but is present if said sequence is at the N-terminus of said conjugate; and X2 and X3 ^{are each} independently any amino acid)
wherein said sequence is present at the C-terminus of the amino acid sequence SLSLSPG, more preferably
a) said heavy chain constant region comprises the sequence SPGSL(FGly')TPSRGS, or b) Z ³⁰ is selected from R, K, H, A, G, L, V, I and P and X ¹ is L , M, S and V, and X2 and X3 ^{are each} independently selected from S, T, A, V, G and C;
Use according to claim 1 . Said modified amino acid residues are located within the light chain constant region of the anti-CD22 antibody, preferably said light chain constant region has formula (II):
X ¹ (FGly') X ² Z ²⁰ X ³ Z ³⁰ (II)
(In the formula,
FGly' is a modified amino acid residue of formula (I);
^Z20 is a proline or alanine residue;
Z ³⁰ is a basic amino acid or an aliphatic amino acid;
X ¹ may or may not be present, and if present may be ^any amino acid, but is present if said sequence is at the N-terminus of said conjugate; and X2 and X3 ^{are each} independently any amino acid)
wherein said sequence is present at the C-terminus of the sequence KVDNAL and/or present at the N-terminus of the sequence QSGNSQ, more preferably
a) said light chain constant region comprises the sequence KVDNAL(FGly')TPSRQSGNSQ, or b) Z ³⁰ is selected from R, K, H, A, G, L, V, I and P and X ¹ is L , M, S and V, and X2 and X3 ^{are each} independently selected from S, T, A, V, G and C;
Use according to claim 1 . Said modified amino acid residue is located within the heavy chain CH1 region of the anti-CD22 antibody, preferably said heavy chain CH1 region has formula (II):
X ¹ (FGly') X ² Z ²⁰ X ³ Z ³⁰ (II)
(In the formula,
FGly' is a modified amino acid residue of formula (I);
^Z20 is a proline or alanine residue;
Z ³⁰ is a basic amino acid or an aliphatic amino acid;
X ¹ may or may not be present, and if present may be ^any amino acid, but is present if said sequence is at the N-terminus of said conjugate; and X2 and X3 ^{are each} independently any amino acid)
wherein said sequence is present at the C-terminus of the amino acid sequence SWNSGA and/or present at the N-terminus of the amino acid sequence GVHTFP, more preferably
a) said heavy chain CH1 region comprises the sequence SWNSGAL(FGly')TPSRGVHTFP, or b) Z ³⁰ is selected from R, K, H, A, G, L, V, I and P and X ¹ is L , M, S and V, and X2 and X3 ^{are each} independently selected from S, T, A, V, G and C;
Use according to claim 1 . The modified amino acid residue is
a) located within the heavy chain CH2 region of the anti-CD22 antibody, or b) located within the heavy chain CH3 region of the anti-CD22 antibody,
Use according to claim 1 .

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